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NATIONAL QUALIFICATIONS CURRICULUM SUPPORT
Biology
Cell and Molecular Biology
Student Monograph
[ADVANCED HIGHER]
Desmond S T Nicholl
University of Paisley
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For Linda, Charlotte, Thomas and Anna
First published by Scottish Consultative Council on the Curriculum 2000
Second impression published by Learning and Teaching Scotland 2001
Electronic version 2001
© Scottish Consultative Council on the Curriculum 2000
Most of this publication may be reproduced for educational purposes by educational
establishments in Scotland provided that no profit accrues at any stage. However, some
pages include items of embedded copyright material. These copyright items are listed
under the acknowledgement below and should not be reproduced without the written
permission of the copyright holder or under licence from the Copyright Licensing Agency
Ltd, 90 Tottenham Court Road, London W1P OLP.
Acknowledgement
Learning and Teaching Scotland gratefully acknowledge this contribution to the Higher
Still support programme for Biology. The author and publisher wish to record their thanks
to Jim Stafford, Biology Editor, of the Higher Still Development Unit.
Thanks are also due to the following for permission to reproduce copyright diagrams and
illustrations: Cambridge University Press for Figures 2.4, 3.10, 4.2, 4.3, and 6.15, all from D
S T Nicholl, Introduction to Genetic Engineering, CUP 1994; Dr A Cawood and Cellmark
Diagnostics for Figures 4.3.3 and 4.3.4; Philip Harris Scientific for Figures 1.1.2, 1.1.3, and
1.1.5, from Cell Structure Resource Pack.
ISBN 1 85955 865 8
Learning and Teaching Scotland
Gardyne Road
Dundee
DD5 1NY
www.LTScotland.com
CONTENTS
Foreword
Section 1:
vii
Structure, function and growth of prokaryotic and
eukaryotic cells
1.1
Features and ultrastructure of prokaryotic and eukaryotic cells
The bacterial cell
Plant cells
Animal cells
1.2
Cell growth and the cell cycle
Mitosis
Control of the cell cycle
Abnormal cell division: cancer cells
8
10
11
12
1.3
Differentiation of cells into tissues and organs
Differential gene expression in development
Segmentation in Drosophila
Embryological development in vertebrates
12
13
13
14
1.4
Cell and tissue culture
Micro-organisms
Mammalian cell culture
15
16
17
1.5
Plant tissue culture
19
Section 2:
Structure and function of cell components
Making and breaking – the molecular architecture
of cells
1
1
3
6
21
2.1
Carbohydrates
The glycosidic bond
Polysaccharides
23
25
26
2.2
Lipids
Triacylglycerols
Phospholipids
Steroids
27
27
28
29
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CONTENTS
2.3
Proteins
Amino acid structure
The peptide bond
Levels of protein structure
Primary (1o) structure
Secondary (2o) structure
Tertiary (3o) structure
Quaternary (4o) structure
Protein motifs and domains
Function of proteins
30
30
31
32
33
33
35
37
37
37
2.4
Nucleic acids
Nucleotide structure
The phosphodiester bond
Polynucleotides and nucleic acid function
39
39
40
40
2.5
Cell membranes
Membrane structure
Function of membrane proteins
43
44
45
2.6
The cytoskeleton
46
Section 3:
Molecular interactions in cell events
3.1
Catalysis
Form and function
The catalytic cycle
Control of enzyme activity
49
50
51
53
3.2
The sodium-potassium pump
57
3.3
Cell signalling
General principles
Extracellular hydrophobic signalling molecules
Extracellular hydrophilic signalling molecules
60
60
61
62
Section 4:
4.1
iv
Applications of DNA technology
The human genome project
Genetic linkage mapping
Physical mapping
DNA sequencing
Comparative genome analysis
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69
71
72
76
78
CONTENTS
4.2
Human therapeutics
Detecting gene disorders
Gene therapy
79
79
83
4.3
Forensic uses
84
4.4
Agriculture
Transgenic plants
Bovine growth hormone
The future?
90
90
92
94
Further reading
95
Glossary
97
BIOLOGY
v
FOREWORD
In this part of the Advanced Higher Biology course we shall be looking
at some aspects of cell and molecular biology. The cell sits at the centre
of our understanding of biology, and has always held a fascination for
biologists of many types. There are those who are interested in how the
components of cells function, and those who see cells as the building
blocks of complex multicellular organisms. Others study the world of
unicellular organisms, or investigate what causes cells to become
cancerous. In modern biology, all of these aspects are studied by
sophisticated methods that enable biologists to probe deeper and
deeper into the events that occur inside cells.
There are three central themes that you should keep in mind as you
study this part of the course:
• Firstly, progress in science requires the development of instruments
and techniques that enable the observations and experiments to be
carried out. Thus the development of cell biology is linked with the
development of microscopes and staining techniques, and the
development of techniques for the fractionation and characterisation
of cell components enabled the field of molecular biology to emerge.
• Secondly, the concept of emergent properties is important in
biology. Thus we can say that the structure and function of biological
molecules depends on their constituent atoms, and in turn the
properties of cells depend on the molecules found inside them.
Likewise the properties of tissues, organs and systems emerge from
the properties of their component cells. Living systems are therefore
organised as a sort of hierarchy, with each level of organisation
building on the level below and enabling new properties to emerge
with the increasing complexity of the system.
• The third theme that is central to cell and molecular biologists is that
of the interlinking of structure and function, regardless of what
level of organisation you consider.
In this book we will begin by looking at the structure, function and
growth of cells. This will set the scene for a look at the biochemical
components of the cell, including some examples of the molecular
interactions that occur inside and between cells. Finally we consider the
applications of DNA technology, which is perhaps the area of modern
biology that holds most promise, yet also raises many ethical problems.
BIOLOGY
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FOREWORD
As we enter the twenty-first century, biology has become a sophisticated,
challenging and exciting discipline, which will continue to have a major
impact on all our lives in the years ahead. By studying the subject at this
advanced level you will be prepared to play your part in the debates that
will undoubtedly take place.
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EUKARYOTIC CELLS
SECTION 1
The study of cell structure was revolutionised by the invention of the
electron microscope. The best optical (light) microscopes can
magnify specimens about 1,500 times under optimal conditions. With
the electron microscope, magnifications of 100,000 times or more are
possible, which enables detailed examination of sub-cellular
components. This structural information, in conjunction with
biochemical studies, has provided a wealth of information about how
cells are constructed and how they function.
1.1 Features and ultrastructure of prokaryotic and
eukaryotic cells
The great variety that is found in different types of cell makes it difficult
to present the ‘average’ cell – there is no such thing! However, there
are things that most cells have in common – the presence of genetic
material, a cell membrane (plasma membrane), and some type of fluidbased matrix (the cytosol) that makes up much of the internal volume
of the cell. We will consider bacterial, plant and animal cells, and try to
illustrate the similarities and differences between them. Major aspects to
consider are the arrangement of DNA in cells, whether or not subcellular organelles are present, the use of membranes within cells, and
the organisation of the cytosol.
The bacterial cell
A generalised diagram of a bacterial cell is shown in Fig. 1.1.1. Bacteria
are prokaryotic, and therefore lack a true membrane-bound nucleus.
The DNA is present as a single circular molecule, usually termed the
bacterial chromosome, although strictly speaking this term should be
reserved for the more complex DNA:protein structures found in
eukaryotic cells. The DNA is highly condensed or ‘packaged’ by coiling
and folding, and this produces a structure known as the nucleoid. Such
packaging is needed because of the length of the DNA molecule – a
typical Escherichia coli cell is about 1 µm diameter × 2 µm length, yet it
contains about 1,400 µm of DNA. Fitting all this into the cell is only
possible because DNA is a very long, thin molecule.
Apart from the nucleoid, there is little internal structure evident in
bacterial cell micrographs apart from a large number of ribosomes,
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essential for protein synthesis. The lack of internal structure means that
the cytosol is effectively the site of all bacterial cell metabolism. This
enables bacteria to adapt very quickly to changing nutritional conditions,
but does mean that the regulation of genetic and metabolic activity has
to be kept under tight control.
Fig. 1.1.1: Diagram of a typical bacterial cell. Not all of the structures
shown may be present in all cells.
Bacteria have cell walls that contain peptidoglycan, which is composed
of linked disaccharide and peptide units. A major method of classifying
bacteria into two groups is the Gram stain, which stains walls according
to how thick the peptidoglycan layer is. Thus we have Gram-positive and
Gram-negative bacteria, depending on whether or not the Gram stain is
taken up by the cell wall. Lying outside the cell wall there may be a
mucilaginous capsule, and there may also be projections from the cell
surface known as pili or fimbriae. Longer flagella may also be present.
Bacterial cells are often considered to be ‘simple’ cells. Whilst this may
be true in terms of the relatively few structural features that are present
within the cell, it is certainly not true when you consider the complex
chemical activity of the cell, all of which has to happen inside the ‘bag’ of
membrane-enclosed cytosol. Most bacterial cells exist as individual
organisms, and need to be metabolically self-contained if they are to
survive.
Cells with a true nucleus and other membrane-bound organelles are
known as eukaryotic cells, and are more complex than bacteria in terms
of structure. Many eukaryotic cells are components of multicellular
organisms, and therefore the requirements of the cell are somewhat
different from those in bacteria.
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Plant cells
A diagram of a typical plant cell is shown in Fig. 1.1.2. Plant cells show
less diversity of form and function than animal cells, and have relatively
rigid cell walls with cellulose as a major component. Thus connections
are required if cell–cell contact is to be achieved. These connecting
structures are the plasmodesmata, which enable a continuous
cytoplasmic link to be formed between cells. The region linking two
adjacent cell walls is known as the middle lamella.
Fig. 1.1.2: Diagram of a cell from a higher plant. Cell sizes vary
considerably in plants, but this would be around 30 × 20 µm.
Distinguishing features of such cells are the cellulose cell wall and the
presence of a central vacuole and chloroplasts. (Copyright Philip Harris
Education. Reproduced with permission.)
Within most plant cells a central vacuole, which looks essentially empty,
occupies a large proportion of the cell volume (up to 90% in some
cases). The vacuole has a much more important role in the life of the
plant cell than its apparent structural simplicity would suggest. The
membrane surrounding the vacuole has an important function in
controlling the movement of substances into and out of the vacuole,
which can act as a storage reservoir for nutrients, waste products,
enzymes and other metabolites. The vacuole is also important in
maintaining cell water relations and thus cell turgor.
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The cytosol of plant cells contains numerous membrane-bound
organelles and other structures that are not present in bacterial cells.
The nucleus is of course the most notable, as this characterises the cell
as being eukaryotic. The nuclear membrane (or nuclear envelope) is a
double membrane structure, with nuclear pores and connections to a
network of membrane-bound channels known as the endoplasmic
reticulum (ER). This may be either Rough ER (ribosomes associated
with the ER membranes) or Smooth ER, which has no ribosomes. The
ribosomes function in the synthesis of proteins that are then
transported via the ER channels to different parts of the cell.
A derivative of the ER system is the Golgi apparatus (sometimes called
the dictyosome in plants). This is named after Camillo Golgi, who first
described it. The function of the Golgi apparatus is to modify and
package materials such as proteins and polysaccharides.
All cells carry out various functions that require energy. Energy
conversion in eukaryotic cells involves two specialist organelles –
chloroplasts and mitochondria. Plants (both algae and higher plants)
are the major primary producers in ecosystems, and have chloroplasts
that localise the reactions of photosynthesis. The chloroplast has a
complex arrangement of membranes arranged in stacks called grana.
These increase the surface area inside the chloroplast and also provide
the membranes on which electron transport proteins are localised.
Chloroplast structure can be seen in Fig. 1.1.3, which shows an electron
micrograph of part of a plant cell.
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Fig. 1.1.3: Electron micrograph of part of a plant cell, showing the
chloroplast. Features labelled are as follows: A – cell wall, B – cell
(plasma) membrane, C – starch grain, D – chloroplast stroma with
ribosomes, E – a granum (stack of thylakoid sacs), F – DNA strands,
G – tonoplast or vacuolar membrane, H – vacuole, I – rough
endoplasmic reticulum, J – cytoplasmic ribosomes, K – chloroplast
envelope, L – intercellular space.
Photographed at 50,000 × magnification. (Copyright A.W. Robards/
Philip Harris Education. Reproduced with permission.)
The light reactions of photosynthesis provide energy by the
photochemical splitting of water, which is used to drive the synthesis of
ATP and NADPH2. These compounds are then available for the dark
(light-independent) reactions, in which carbon dioxide is reduced to
carbohydrate.
Energy capture in photosynthesis, and the consequent fixing of carbon
dioxide into sugar, is the single most important set of reactions in
biological systems. Without photosynthesis, there would be no
carbohydrate available to be metabolised in respiration, and thus cells
could not survive. In aerobic organisms glucose can be completely
oxidised to carbon dioxide and water by the processes of glycolysis and
the tricarboxylic acid (Krebs) cycle. The major energy yield in this
process occurs during the re-oxidation of NADH2 in mitochondria.
These are in some ways similar to chloroplasts in that they have a folded
membrane arrangement on which the electron transport proteins are
arranged to enable ATP synthesis to be driven by proton pumping
mechanisms.
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STRUCTURE, FUNCTION AND GROWTH OF PROKARYOTIC AND
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Chloroplasts and mitochondria are essential parts of the biological
energy chain, and both illustrate how membranes can be used within
cell organelles to increase the area available for the localisation of
biochemical reactions. A comparison of the structure of chloroplasts and
mitochondria is shown in Fig. 1.1.4.
Fig. 1.1.4: Comparison of the internal structure of mitochondria and
chloroplasts. The use of membranes to increase the surface area/volume
ratio, and the presence of DNA, are common features of these two
organelles.
Animal cells
We have already met most of the components of animal cells, as they are
similar to those found in plant cells. There are two notable exceptions –
animal cells do not have chloroplasts or cell walls. In addition, any
vacuoles present are usually very small (and transient) when compared
to the plant cell vacuole. Animal cells show a much greater variation in
structure and function than plant cells. This variation is however
achieved by using the ‘standard’ set of cell components in different
ways, depending on the type of cell.
A diagram of an animal cell is shown in Fig. 1.1.5. In addition to the
main components such as the nucleus, Golgi apparatus, endoplasmic
reticulum and mitochondria, animal cells may have microvilli on the
cell surface. They also have centrioles, which assist in the organisation
of spindle fibres during cell division. Some components of animal cells
as revealed by electron microscopy are shown in Fig. 1.1.6.
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Fig. 1.1.5: Diagram of an animal cell. This cell would be approximately
20 µm in diameter. Note the lack of a cell wall, vacuole and
chloroplasts. (Copyright Philip Harris Education. Reproduced with
permission.)
Fig. 1.1.6: Electron micrograph
of cells in rat liver. Features
labelled are as follows: A –
rough endoplasmic reticulum,
B – mitochondria,
C – mitochondrial envelope
(highlighted for clarity),
D – nuclear envelope,
E – nucleus (nucleoplasm),
F – part of the Golgi apparatus,
G – cell membrane separating
two adjacent cells, H –
glycogen granules.
Photographed at 25,000 ×
magnification. (Copyright A.W.
Robards/Philip Harris
Education. Reproduced with
permission.)
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STRUCTURE, FUNCTION AND GROWTH OF PROKARYOTIC AND
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In looking at plant and animal cells, we have seen that the use of
membranes to provide internal structure is a central theme. The
intracellular arrangement of membranes is often called the
endomembrane system, and can be considered as a way of organising
the cytosol so that the complex metabolism required for eukaryotic cell
function can be controlled and regulated.
In addition to the main subcellular structures already described for plant
and animal cells, there are other organelles and structures found in
eukaryotic cells. These include a variety of membrane-bound vesicles
such as microbodies (also called peroxisomes) and lysosomes. The
function of these vesicles is often to isolate specific reactions which
might otherwise be harmful to the cell if they were not kept separate
from the rest of the cytosol. Other structures of importance are the
elements of the cytoskeleton, namely microtubules, intermediate
filaments and microfilaments. We will consider the cytoskeleton in
more detail elsewhere in this book.
The organisation of DNA within the nucleus of a eukaryotic cell is more
complex than the ‘naked’ DNA found in prokaryotes. Eukaryotes have
multiple chromosomes, and the packaging problem that is evident even
in bacteria is more pronounced. In human cells, some 2m of DNA must
be packed into a nucleus that is about 5µm in diameter. This is achieved
by coiling the DNA around nucleosomes, which are made up of histone
proteins. More extensive coiling of the nucleosome chain is required,
particularly during cell division, and this produces the tightly packed
structures that we recognise as eukaryotic chromosomes.
1.2 Cell growth and the cell cycle
The cell theory states that cells can only arise by the division of existing
cells. Cell growth and division is therefore a critical process in the life of
cells and organisms. The reproduction of cells, from when the cell is
produced by division of the mother cell until the new cell itself divides,
is known as the cell cycle. The length of this cycle varies depending on
the cell type. Bacterial cells can divide every 20 minutes when growth
conditions are favourable, whereas human liver cells only divide about
once a year. Mammalian cells in tissue culture have a cell cycle time of
about 20 hours; frog embryo cells divide much more frequently with a
cycle time of around 30 minutes.
Despite the variation in the duration of the cell cycle, there are certain
basic requirements for cell division. If two new cells are to be produced,
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clearly the amount of cell material must double if the daughter cells are
to remain the same size as the parent. This is particularly critical for the
DNA of the cell, which must be copied exactly if the genetic integrity of
the cell is to be maintained. This is achieved by the process of DNA
replication, which is an important marker during the cell cycle.
How has the cell cycle been studied? If you were to look at a mammalian
cell under the microscope for a complete cell cycle, you would see very
little evidence of cytological changes until the last hour of the process,
when suddenly there is a flurry of activity as the chromosomes move
apart and two new cells are formed. Thus early descriptions of the cycle
separated the process into two parts, called interphase and division.
The division process itself is more accurately called mitosis or the M
phase of the cycle. Mitosis refers to the separation of the chromosomes,
and this is followed by division of the cell during cytokinesis (CK).
Interphase is sometimes called the resting phase, but in biochemical
terms this is in fact a very active period of growth. A more accurate
description of the cell cycle splits interphase into three parts. The
period of DNA replication is known as the synthesis or S phase, and
there are ‘gaps’ known as G 1 and G2. As our knowledge has increased, it
has become clear that ‘gap’ is perhaps not the best way to describe these
two periods, as this suggests little activity, as does the term interphase. A
diagram of the cell cycle is shown in Fig. 1.2.1.
Fig. 1.2.1: The cell cycle.
Interphase is made up of phases
G1, S and G2. During S phase
the DNA is replicated, although
no cytological changes are
distinguished in the light
microscope. The cell grows
during interphase and enters
the M phase (mitosis). The four
sub-stages of M phase are
prophase (P), metaphase (M),
anaphase (A) and telophase
(T). This is followed by
cytokinesis (CK) which
generates two new daughter
cells, each of which enters G1 to
begin the cycle again. For
animal cells in tissue culture,
cycle duration is around 20
hours.
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Mitosis
The actual division of the cell requires a series of co-ordinated events
that separate the replicated chromosomes and then split the cell into
two, each half having received a complete set of chromosomes. After
DNA replication the chromosomes are composed of two chromatids,
held together at the centromere. Following separation during mitosis,
the newly separated sister chromatids are known as daughter
chromosomes.
Although mitosis is a dynamic process, there are four stages that can be
recognised easily in the light microscope. These are prophase,
metaphase, anaphase and telophase. Movement of chromosomes is
achieved by spindle fibres, which are microtubules. These are
composed of alternating dimers of α and β tubulin (a protein). The
spindle begins to form at prophase, and is organised by spindle poles or
centrosomes at the two poles of the cell. During mitosis the spindle
serves to guide the daughter chromosomes and pull them apart to
opposite ends of the cell. Fig. 1.2.2 shows the events of mitosis in onion
cells.
Fig. 1.2.2: Mitosis in onion cells (Allium sp.). Photographs represent
cells seen under the light microscope at 100 × magnification. The
diagram and explanation describe the events in each stage.
Due to the structural differences between plant and animal cells,
cytokinesis is achieved by different mechanisms in these two cell types.
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In animal cells, constriction of the cytoplasm by a contractile ring of
actin and myosin produces a cleavage furrow, which essentially pinches
the cell into two pieces. In plants the cell wall makes this impossible,
and cytokinesis is achieved by the formation of a new cell wall to
separate the daughter cells.
Control of the cell cycle
What controls cell division? This is a question that has occupied
biologists for many years, and a wide variety of different organisms and
cell types has been used to try to answer some of the main questions.
Obviously it is important that DNA replication is complete before
mitosis, and that the cell mass has increased sufficiently to enable two
daughter cells to be formed. Also, the division processes of mitosis and
cytokinesis have to be controlled in both temporal and spatial terms if
success is to be achieved. In multicellular eukaryotes, additional
constraints on cell division are required if cell numbers are to be
controlled. To divide, these cells require specific growth factors, of
which over 50 types have been isolated. In the absence of enough
growth factor, cells stop at the G1 checkpoint and enter a non-growing
phase called G0.
Our current understanding of cell cycle control is that there is a central
mechanism that is used to assess the status of the cell as it progresses
through the cycle. This mechanism works through a series of three main
checkpoints:
• G1 checkpoint – towards the end of G 1 phase, cell size is assessed. If
sufficiently large to allow division, the cell enters S phase and DNA
replication begins.
• G2 checkpoint – the success of DNA replication is monitored, and if all
is well entry into mitosis is triggered.
• M checkpoint – this occurs during metaphase and triggers the exit
from mitosis and cytokinesis, and entry into the next G1 phase in the
daughter cells.
The molecular mechanisms that control the cell cycle involve the
interactions of many different genes and proteins to ‘trigger’ the events
of the cell cycle in their proper sequence. For example, a critical part of
cell division is the entry into mitosis. This is triggered by a complex
called mitosis promoting factor (MPF), which in turn is controlled by
other intracellular cell cycle signals.
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Abnormal cell division: cancer cells
Cancer cells are those in which the normal control of cell proliferation
has been lost, enabling these cells to form tumours. Although often not
the only defective function, this loss of cell cycle control is a major
feature of cancer cells. There are two main classes of genes that can
generate abnormal cell division. Proliferation genes encode proteins
that promote cell division, and any over-expression of these genes could
result in excessive cell division. Genes in this category are called
oncogenes when mutated (proto-oncogenes when normal). The
second class of cancer-causing genes are known as antiproliferation
genes, which normally help to restrict cell division by acting at cell cycle
checkpoints. These genes are sometimes called tumour-suppressor
genes. In diploid cells, only one copy of a proto-oncogene has to
mutate into an oncogene to cause a problem, whereas both copies of a
tumour-suppressor gene would have to become abnormal. Thus
mutations in proto-oncogenes are dominant, those in tumoursuppressor genes are recessive. Our knowledge of how cancer cells arise
has developed greatly over the past few years, and many types of protooncogene and tumour-suppressor gene have been identified.
1.3 Differentiation of cells into tissues and organs
Although the cell is essentially a complete living system in its own right,
in multicellular organisms cells are organised into tissues and systems
for specific functions such as support, movement, nutrition, coordination and control. Much useful information about tissues and
systems has been obtained by studying the final product – liver, kidney,
nerves, glands and so on – but the key question is concerned with how
these tissues and systems arise in the developing embryo. This is the
area of developmental biology. When scientists began to isolate and
study genes, a logical step was to try to understand how genes function
in the control of developmental processes. Thus the field of
developmental genetics has emerged as one of the central areas of
modern biology. Despite great advances in our knowledge,
development remains one of the most complex and astonishing
branches of science.
One way of looking at complex processes is to study simple organisms,
to see if any functions correlate with those in higher organisms.
Although there are not many developmental processes evident in
bacteria, some aspects of how genes are expressed have become clearer
by looking at adaptive responses such as expression of the lac operon.
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Multicellular plants provide relatively well-ordered systems, with much
simpler developmental patterns than animals, yet development in plants
is not as well understood as in animals.
Development in animals begins with a single fertilised egg undergoing
successive divisions. Cell proliferation is of course needed to make up
the cell number required as the embryo develops – an adult human has
more than 1,000,000,000,000 cells! However, all these cells divide
mitotically, and so have identical genomes. So how do different cells
arise? And how do they end up in the correct place as the embryo
grows?
Differential gene expression in development
To generate different cells from the same genetic information, there
must be some sort of control over gene expression. This control can be
both temporal (different genes expressed at certain specific times in
development) and spatial (cells in different places in the embryo
expressing different genes). Development is not a fixed series of genetic
events, but rather a ‘conversation’ between cells as the embryo
develops. This ‘conversation’ involves gene products, which influence
cellular events, which in turn create patterns due to cell movement and
differentiation. Unravelling all this complexity is an immense task, but it
is becoming clear that similar processes operate in all animals. Studies
on the fruit fly Drosophila melanogaster have enabled researchers to
gain an almost complete understanding of how genes influence
development in this organism. We will look at some examples to
illustrate.
Segmentation in Drosophila
The body of a mature Drosophila has 17 segments. These are established
early in development by the action of several genes in a hierarchical
sequence where the action or effect of one set of genes depends on the
effects of genes earlier in the developmental sequence. In Drosophila,
the first set of genes produce gradients of concentration of gene
product which determine the anterior/posterior and dorsal/ventral axes.
The next set of genes respond to these gradients to divide the embryo
into four main segments. The action of yet other genes further subdivides the embryo, and then sets up the final segmentation pattern. At
this time regulatory (homeotic) genes control structural genes to
determine the final fate of each of the segments by specifying the type of
appendages and other structures that are specific for each segment.
Gene action in Drosophila pattern formation is summarised in Fig. 1.3.1.
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Fig. 1.3.1: Differential gene expression during pattern formation in
Drosophila. The developing embryo is divided into smaller and smaller
segments by the sequential action of different sets of genes.
Embryological development in vertebrates
In addition to differential gene expression, other factors are important
in development of the animal embryo. In vertebrates, the fertilised egg
divides to produce a ball of cells called the blastula, which then folds in
on itself during the process of gastrulation. This sets up the major body
plan of the embryo, and by a ‘conversation’ process similar to the
Drosophila example, cells interact with each other, move and
differentiate to give progressively higher degrees of order in the
embryo. A schematic representation of this series of events in the mouse
is shown in Fig. 1.3.2.
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EUKARYOTIC CELLS
Fig. 1.3.2: From egg to organism – the sequence of events in the
development of the mouse embryo. Vertebrate development involves a
series of complex interactions involving differential gene expression,
cell signalling, and cell movement.
Developmental biology is a complex and exciting area of biology, that
constantly provides new insights into how genes function to provide
form and structure in the developing organism. It is clear that
development involves molecular ‘conversations’ between cells, and that
it is therefore a dynamic and interactive process rather than a simple
reading of the genetic instructions.
1.4 Cell and tissue culture
The ability to grow cells in culture is essential for both pure and applied
aspects of biology. The applications of cell culture are many and varied –
perhaps growing bacterial cells for a basic gene manipulation procedure,
culturing mammalian cells for cancer studies, or producing new plants
by using tissue culture techniques. Although there are marked
differences in the techniques and growth media used with different cell
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EUKARYOTIC CELLS
types, there are certain things that are required for all cell culture
procedures:
• a source of the cell material – either freshly prepared, or from a stock
of the cell line or bacterial culture.
• a suitable container for cell growth – a simple flask may be sufficient,
or a sophisticated fermenter with computer-controlled monitoring of
culture conditions might be needed.
• a growth medium that provides all the required nutrients.
• opportunity for gas exchange (chiefly oxygen and carbon dioxide).
• control of factors such as temperature and pH.
• a method for measuring cell growth – this might involve counting cell
numbers in the culture, or measuring the optical density in a
spectrophotometer.
• avoidance of contamination of the culture with unwanted microorganisms.
Let’s look at some aspects of cell and tissue culture using microorganisms and mammalian cell lines as examples.
Micro-organisms
Microbes inhabit many diverse ecological niches, and we might therefore
expect that they should be adaptable and relatively easy to culture in the
laboratory on a small scale (up to a few litres of culture). In addition,
many types of microbe are used in the biotechnology industry in
processes for the production of useful compounds. Industrial scale
operations can involve culture volumes of up to tens or even hundreds
of thousands of litres. The term fermentation is often used to describe
any micro-organism growth procedure, although technically the term
refers to anaerobic growth only.
Unicellular algae have few requirements, and can be grown in simple
mineral salts media. Bubbling with air (often enriched with carbon
dioxide) increases the growth rate and yield of these photoautotrophs.
Bacteria and yeasts need more complex media, as they are
heterotrophic and therefore need an organic carbon source and other
compounds such as amino acids.
Micro-organisms can be grown in batch culture, where a culture is
grown without dilution until maximum attainable density is reached.
Batch cultures under appropriate conditions show a period of
exponential growth, but this becomes limited by nutrient availability or
cell density effects. Cultures then enter a stationary phase and
eventually die if not sub-cultured.
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Exponential growth can be maintained in continuous culture if
conditions are altered as growth continues. This can be achieved by
continual addition of fresh medium and removal of an equal volume of
the culture.
Mammalian cell culture
Animal cells are fragile and require more carefully controlled conditions
than microbial cells if growth is to be maintained. Growth media are
more complex, although the basic requirements are similar to those for
bacterial cells. The minimum growth medium recipe consists of a
balanced salt solution with amino acids, vitamins and glucose. Useful
additions are a pH indicator and antibiotics to prevent bacterial growth,
but the main additional requirement is for animal serum such as foetal
bovine serum (FBS). This is difficult to define chemically, but many of
the components appear to be essential for animal cell proliferation.
Animal cell technologists have been trying to establish defined media for
cell culture, but to date only a few cell types are supported by serumfree media. Growth media containing 5–10% FBS are often used for cell
culture.
Cells can be obtained by treating tissue samples with a proteolytic
enzyme (such as trypsin) to separate cells from each other. This gives a
primary cell culture, from which secondary cultures can be derived.
However, one drawback is that normal cells only divide a finite number
of times before they die, and thus long-term culturing of primary cell
cultures is difficult. The most common cell lines used today have either
been derived from tumours or have been transformed to produce
immortalised cell lines. These cell lines are neoplastic, that is they
produce cancers if transplanted into animals. Some common animal cell
lines are shown in Table 1.4.1.
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Table 1.4.1: The origin and properties of some commonly used animal
cell lines. Cells generally grow as monolayers in tissue culture flasks.
Those that are able to grow in suspension culture conditions are
indicated.
Cell line
Species of
origin
Tissue of
origin
Cell
morphology
Growth in
suspension?
3T3
Mouse
Connective
Fibroblast
No
CHO
Chinese hamster
Ovary
Epithelial
Yes
BHK21
Syrian hamster
Kidney
Fibroblast
Yes
HeLa
Human
Cervical
carcinoma
Epithelial
Yes
Cells in tissue culture generally grow as a monolayer adhering to the
bottom of the plastic flask used as the culture vessel. Animal cells
growing in culture are shown in Fig. 1.4.2. When the cells cover the
available surface, they are said to be confluent, and proliferation stops
until cells are sub-cultured into fresh medium.
Fig. 1.4.2: Animal cells growing in
tissue culture. The cells form a flat
sheet or monolayer on the bottom of
the culture flask. One cell is outlined
with a white border, showing the
boundary of the cell (formed by the
cell membrane) and the nucleus.
(Courtesy of Dr Dajiang Li.)
One of the main advantages of growing cells in culture is that they can
be selected and cloned – that is, a culture of identical cells can be
derived from one isolated cell. This has been useful for the isolation of
mutant cell lines, which can be used to understand normal cell growth
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processes and may also be useful in biotechnological applications. In
addition, cell lines can be fused to produce new hybrid cells which
often have useful characteristics.
1.5 Plant tissue culture
Plant cells can be grown in culture in a similar way to animal cells,
although the growth requirements are simpler and defined media can be
prepared more easily. One particularly useful trait of plant cells is their
ability to regenerate complete plants under appropriate conditions. In
theory, all somatic cells from multicellular organisms have this potential,
as they have the same genome – this is called nuclear totipotency.
However, animals are much too complex to regenerate directly from
somatic cells, and thus the applications of regeneration have been
restricted to plants. Many commercially important ornamental plants are
propagated in this way. Tissue culture is also important in the
production of pathogen-free plants.
Small pieces of plant tissue (explants) can be taken and grown on a
medium containing plant growth regulators (plant hormones) such as
auxin and cytokinin. Cell proliferation produces an undifferentiated
mass of cells known as a callus. By sub-culturing and changing the
balance of growth regulators, the callus tissue can be coaxed into
differentiating into shoots and roots, and can be planted out to
regenerate complete plants. Callus tissue with developing shoots can be
seen in Fig. 1.5.1.
Fig. 1.5.1: Plant cell culture. This shows cells of potato growing on a
petri dish in callus form. The cultures were derived from protoplasts of
the potato cells. The formation of shoot tissue from some of the calli can
be seen. (Courtesy of Dr Y Hamidoghli)
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Hybrid plant cells can be generated using a technique known as
protoplast fusion. A protoplast is a plant cell with the cell wall removed
enzymatically. This enables cells to fuse together if conditions are
favourable, and thus new combinations of cells can be produced.
Protoplasts are also useful for gene manipulation procedures in plants,
as the lack of a cell wall means that recombinant DNA can be readily
taken up by the cell. Protoplast fusion is shown in Fig. 1.5.2.
Fig. 1.5.2: Fusion of protoplasts from two different species of potato.
One unfused protoplast (U) is shown, lying on top of another. Compare
this with the two heterokaryons shown (H). These are produced when
two protoplasts join together during fusion. (Courtesy of Dr Y
Hamidoghli)
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SECTION 2
Information about the ultrastructure of cell components, obtained by
using the electron microscope, has provided much useful detail about
how cells are organised. However, to get a full picture of how cells
work, it is necessary to examine the biochemistry of the various
molecules and structures found inside cells. The development of
biochemical and molecular analysis has followed a similar path to that
for microscopy, with more detailed information becoming available as
more sophisticated techniques were developed.
Much of the structural information about biological molecules has come
from an understanding of their fundamental chemistry. This approach
has been extended by studying how these molecules function in their
biological roles, which has enabled the elucidation of the many
metabolic pathways that are required for cells to function. Modern
developments in molecular genetics can now provide information about
how genes work at the molecular level, and thus in many cases the
complete picture of how a particular molecular system functions at the
cellular level is becoming clear. Many of the important discoveries of
recent years in areas such as developmental biology and cancer have
come from this approach, which is sometimes called molecular cell
biology.
In this section we consider the main groups of molecules that are found
in cells – carbohydrates, lipids, proteins and nucleic acids. We then
consider two important cellular systems; cell membranes, and the
cytoskeleton.
Making and breaking – the molecular architecture of cells
One central theme runs throughout biochemistry – the making and
breaking of chemical bonds. Living systems are composed of a limited
number of elements, with carbon, hydrogen, nitrogen, oxygen,
phosphorus and sulphur (CHNOPS) making up around 99% of their
mass. The carbon atom is of central importance, as it can form four
covalent bonds with other atoms. This enables a variety of complex
molecules to be constructed. In addition to the chemistry of carbon
itself, there are many important functional groups associated with
biological molecules. Some of these are shown in Fig. 2.1.
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STRUCTURE AND FUNCTION OF CELL COMPONENTS
Fig. 2.1: Some functional
chemical groups that are
important in biological
systems. Groups such as the
carboxyl, amino and
phosphate groups can be
ionised, and thus carry a
charge at neutral pH. The
ionised forms of these groups
are shown alongside the nonionised version.
Many biologically important molecules are polymers, composed of
smaller units called monomers linked together. Two monomers are
joined by removing the elements of water. This reaction is a
dehydration synthesis (a specific type of condensation reaction) and
can be reversed by adding back the elements of water by hydrolysis.
This feature of being able to construct and de-construct large molecules
or macromolecules is one of the most important aspects of cell
metabolism (see Fig. 2.2).
Fig. 2.2: The monomer/polymer cycle. Monomeric units can be joined
together by dehydration to give polymers. Hydrolysis reverses this and
regenerates the monomers. Cyclical polymerisation/depolymerisation
like this is important in many cellular processes.
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Making and breaking chemical bonds involves energy. A broad
generalisation is that making more complex structures from simpler
precursors requires energy (anabolic or biosynthetic reactions)
whereas breaking bonds releases energy (catabolic reactions). Where
there is little overall energy change, reactions are said to be reversible.
The metabolism of a cell involves a complex mixture of these three types
of reaction, all interacting with and responding to different
concentrations of reactants and products, and the whole lot having to be
very tightly controlled if energy chaos is to be avoided. The wonder of
metabolism is that it works at all!
In considering the structure of biological molecules, it is important to
remember that these are three-dimensional arrangements of atoms. In
fact, all the reactions in cells depend on the shapes of molecules, so this
is a critical point. Whilst it is often difficult to represent 3-D structures in
2-D form (i.e. diagrams), there are a number of conventions that can be
used. We will come across some of these as we look at the molecules
and macromolecules of the cell.
2.1 Carbohydrates
The carbohydrates are composed of carbon, hydrogen and oxygen in
the ratio 1:2:1, giving a molecular formula of (CH2O)n for most simple
carbohydrates. These are the monosaccharides or ‘single sugars’. There
is considerable variation in monosaccharide structure, based on the
number of carbon atoms and the arrangement of the hydrogen and
oxygen atoms attached to them. We will examine the most common
monosaccharide that is of central importance in biological systems –
glucose.
Glucose (C6H12O6) can be defined by its six carbons (making it a hexose
sugar) and by the arrangement of the carbonyl (C=O) group at the
terminus of the molecule. A different arrangement of the carbonyl group
gives a different spatial arrangement of the atoms of a hexose sugar. All
these variations of the same C6H12O6 formula, known as isomers, make
carbohydrate structure a complex topic.
The simplest representation of glucose is the straight-chain form, shown
in Fig. 2.1.1. By convention, if the OH group on carbon 5 (C5) projects
to the right, the form is the D-form; if to the left, the L-form. Most sugars
used in biological systems are the D-forms. Thus the representation in
Fig. 2.1.1 is designated D-glucose. However, in solution glucose adopts a
predominantly cyclical form, where C1 and C5 are linked through the
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STRUCTURE AND FUNCTION OF CELL COMPONENTS
oxygen atom to give a ring structure. Depending on the arrangement of
the hydroxyl group on C1, this generates two further variations known
as the alpha and beta structures. In α-D-glucose the hydroxyl group
attached to C1 is below the plane of the ring, in β-D-glucose the hydroxyl
group is above the plane of the ring. In solution the equilibrium
proportions of the three forms are approximately 38%
α-D-glucose and 62% β-D-glucose, with at any given time only about
0.02% straight-chain form, with some other minor derivatives possible.
Fig. 2.1.1: The straight-chain form of glucose. The carbon atoms are
numbered 1–6, with the carbonyl group at C1.
As if trying to make sense of carbohydrate structure wasn’t difficult
enough, 3-dimensional representations give a better idea of what the
molecule actually looks like. Fig. 2.1.2(a) shows a full representation of
the structure of α-D-glucose, with Fig. 2.1.2(b) showing β-D-glucose in the
standard shorthand version that is commonly used when drawing
carbohydrate structures.
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Fig. 2.1.2: Haworth projections of the glucose molecule. (a) shows
α-D-glucose with the carbons and hydrogens shown. The thicker lines
show the 3-D effect. (b) shows β-D-glucose in the simplified form. Here,
the carbons and hydrogens are not shown (unmarked corners represent
carbon, unmarked line ends hydrogen). In addition, the 3-D line
thickenings are not shown. It is assumed that the ring projects with the
bottom edge towards the viewer. The OH groups that define the α and β
forms are shaded.
The glycosidic bond
Two monosaccharides can be linked by a dehydration synthesis to give a
disaccharide. These are defined by the component momomers and by
the way in which the bond is arranged. If we consider two glucose
monomers, the bond will be between the C1 of one molecule and the
C4 of the other, giving either an α(1,4) linkage (maltose) or a β(1,4)
linkage (cellobiose). These disaccharides are shown in Fig. 2.1.3. Other
disaccharides include common table sugar sucrose (glucose and
fructose), and the milk sugar lactose (glucose and galactose).
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STRUCTURE AND FUNCTION OF CELL COMPONENTS
Fig. 2.1.3: Disaccharide structure. Two glucose monomers can be joined
by an α(1,4) linkage to give maltose. If the linkage is a β(1,4)
arrangement, this give cellobiose The H/OH configuration on the C1 on
the right hand side of the diagrams is not specified, as this can exist in
either the α or β arrangement.
Polysaccharides
Joining more monomeric units together produces larger polymers. If
the repeating units are the same, we have a homopolymer, whereas
different subunits give a heteropolymer. Long chains of simple sugars
give the polysaccharides. There are many types of polysaccharide,
including starch, glycogen and cellulose. These are all homopolymers
made from glucose monomers linked together, but have markedly
different properties and functions. This again illustrates how diversity of
form and function can be generated by relatively simple variations in the
chemistry of the molecules. The structure of these three examples is
shown in Fig. 2.1.4.
Fig. 2.1.4: Polysaccharide structures
found in starch (amylose, helical
arrangement), glycogen (branched)
and cellulose (parallel chain
arrangement). Each of these is
composed of a chain (or chains) of
glucose monomers.
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Glycogen (animals) and starch (plants) are storage polysaccharides,
readily hydrolysed to release the monomers for catabolic breakdown to
provide energy. Cellulose is a much tougher arrangement of fibres that
is ideally suited to its structural role in plants. It is the most abundant
organic material on earth, yet most animals lack the enzyme cellulase
that is needed to break it down into its component monomers. Other
polysaccharides include chitin, a homopolysaccharide found in fungal
cell walls and insect exoskeletons, and the glycosaminoglycans, which
are heteropolysaccharides found in skin and connective tissues in
vertebrates.
2.2 Lipids
Lipids are important in cell membrane structure, and also as hormones
and energy storage molecules. The common defining feature of lipids is
that they are insoluble in water. Fats and oils are familiar lipids that we
use every day, the distinction being a rather arbitrary one of the physical
state of the molecule at room temperature.
Although lipids are certainly smaller molecules than the large
polysaccharides, proteins and nucleic acids, they are generally classed as
one of the four groups of macromolecules. Three types of lipid are of
particular importance in cells: triacylglycerols (or triglycerides),
phospholipids and steroids.
Triacylglycerols
The constituents of triacylglycerols are a glycerol ‘backbone’ and fatty
acids. Glycerol is a 3-carbon alcohol; fatty acids are hydrocarbon chains
ending with a carboxyl group. If all the available bonds are occupied by
hydrogens, the fatty acid is said to be saturated. If there are carboncarbon double bonds in the molecule, this gives an unsaturated fatty
acid. One structural consequence of this is that saturated fats pack
closely together and tend to be solid, whereas in unsaturated fats kinks
are introduced and the fatty acid chains do not fit together closely. This
generally means that unsaturated fats are oils rather than hard fats. Most
animal fats are saturated, those from plants tend to be unsaturated.
Some common fatty acids are shown in Table 2.2.1.
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Table 2.2.1: Some common fatty acids found in lipids. Saturated fatty
acids have no C=C double bonds. Oleic acid has one C=C double bond,
linoleic acid has 3, and is therefore polyunsaturated.
Fatty acid
No. of
carbons
Saturated/
unsaturated
Structure
palmitic acid
16
saturated
CH3(CH2)14COOH
stearic acid
18
saturated
CH3(CH2)16COOH
oleic acid
18
unsaturated
CH3(CH2)7CH=CH(CH2)7COOH
linoleic acid
18
polyunsaturated
CH3(CH2CH=CH)3(CH2)7COOH
Glycerol and fatty acids are joined together by dehydration
(condensation) synthesis reactions between the hydroxyl and carboxyl
groups, generating a triacylglycerol (triglyceride). This is shown in Fig.
2.2.2.
Fig. 2.2.2: Triglyceride structure. The glycerol molecule acts as the
‘backbone’ to which three fatty acids are attached by ester linkages. This
gives a triacylglycerol or triglyceride. The properties of triacylglycerols
are determined by the properties of the fatty acids
Phospholipids
An important variant of the triacylglycerol structure is where one of the
fatty acids is replaced by a phosphate group, which often has other
groups attached. Usually one fatty acid is saturated, and one is
unsaturated. The most abundant phospholipid in animal tisue is
phosphatidylcholine (Fig. 2.2.3).
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Fig. 2.2.3: Phosphatidylcholine. This phospholipid has two fatty acid
tails, one saturated (S) and one unsaturated (US). The polar head
region is a choline/phosphate group linked to the glycerol backbone.
As phospholipids have a non-polar fatty acid ‘tail’ and a polar ‘head’,
they are hydrophilic (water-loving) at the head and hydrophobic
(water-hating) at the tail. This is a critical property in that it enables
phospholipids to form bilayers, as shown in Fig. 2.2.4. As we shall see
later, this is important in membrane structure.
Fig. 2.2.4: Phospholipids can form bilayers, with the polar heads on the
outer (hydrophilic) surface, and the fatty acid tails forming a
hydrophobic inner region.
Steroids
These lipids have a markedly different structure to that found in the
glycerol-based triglycerides and phospholipids. Steroids are based on a
four-ring structure, with associated side chain variations. The best
known example of a steroid is cholesterol, which is found in cell
membranes. Other steroids such as testosterone are hormones. The
structure of cholesterol is shown in Fig. 2.2.5.
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STRUCTURE AND FUNCTION OF CELL COMPONENTS
Fig. 2.2.5: The structure of cholesterol is based on a four-ring structure.
Not all the atoms are labelled. Testosterone and other steroid hormones
are similar, with different attached groups defining their chemical
properties.
2.3 Proteins
Protein molecules are heteropolymers made up of amino acids, of
which there are 20 that are used in proteins. Variation in the length of
the amino acid chain, and the order of the individual amino acids in it,
theoretically enables an essentially unlimited variety of proteins to be
constructed. This makes proteins the most diverse group of
macromolecules in the cell, with many different roles to play in both
structural and functional terms.
Amino acid structure
Amino acids are characterised by the amino (HN2) group and the
carboxylic acid (COOH) group. These are attached to the central or
alpha carbon atom, which also carries a hydrogen atom and the variable
part of the molecule (the R-group). Like carbohydrates, amino acids
show isomerism, existing in both the D- and L-forms. The L-form is found
exclusively in proteins. At neutral pH amino acids exist in the ionised
form, although the charges on the amino and carboxylic acid groups
disappear when the monomers are joined together. The simplest amino
acid is glycine, which has a hydrogen atom as the R-group. A methyl
(CH3) group gives alanine, shown in Fig. 2.3.1.
Fig. 2.3.1: Amino acid structure. Amino acids have a hydrogen, and
amino group and a carboxyl group attached to the central α-carbon
atom. The fourth position is a variable side-chain or R-group. In this
example the R-group is a methyl group, giving the amino acid alanine.
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As the variable part of amino acid structure, it is the R-group that gives
each amino acid unique chemical properties. Interactions between Rgroups also specify the particular shape that the protein has, and
determine its overall properties. R-groups can be classed as acidic,
basic, uncharged polar, and nonpolar. Some examples of the types of
side chain found in these classes are shown in Table 2.3.2.
Table 2.3.2: Types of R-group found in amino acids. The number of
amino acids (total 20) in each class is shown. The three-letter and
single-letter abbreviations are shown as Asp / D etc.
Class/No.
amino acid
amino acids
abbreviation R-group
Acidic
(2)
Asp / D
aspartic acid
-CH 2COOH (ionises to COO -–)
glutamic acid Glu / E
-CH 2CH 2COOH (ionises to COO -–)
Basic
(3)
lysine
Lys / K
-(CH 2) 4NH2 (ionises to NH 3 + )
Uncharged
polar
(5)
serine
Ser / S
-CH2OH
asparagine
Asn / N
-CH2C=O
NH2 (uncharged but polar)
glycine
Gly / G
-H
alanine
Ala / A
-CH3
cysteine
Cys / C
-CH2SH
Nonpolar
(10)
The peptide bond
Proteins are made by joining amino acids together by an amide linkage
called the peptide bond. A chain of amino acids is therefore known as a
polypeptide. The peptide bond is formed by a dehydration synthesis
reaction between the carboxylic acid group of one amino acid and the
amino group of the next, as shown in Fig. 2.3.3. Amino acids joined
together in this way are called residues. Although the peptide bond
itself is a planar (flat) structure which does not allow rotation around
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the C-N bond, the single bonds on either side of the peptide bond do
allow rotation of the residues, which makes polypeptide chains very
flexible structures. This is important in determining the way in which
chains of amino acids can fold to generate the highly ordered structures
found in complex proteins.
Fig. 2.3.3: The peptide bond links amino acids together by a
dehydration synthesis between the carboxyl and amino groups of two
different amino acid monomers. This gives the C-N linkage that is
characteristic of the planar (flat) peptide bond.
Levels of protein structure
No group of biochemical molecules illustrates the concept of emergent
properties better than the proteins. From the constituent atoms of the
amino acids up to the final form of a large protein macromolecule,
progressively more complex structural organisation enables specific
form and function to be generated. To make sense of this complexity,
protein chemists recognise four levels of protein structure, beginning
with the sequence of amino acids in the polypeptide chain.
Chemical bonding is obviously critical in determining protein shape, and
different types of bonds are important at different levels. The covalent
peptide bond that links amino acid residues together is a very strong
bond. In higher-order protein structure weaker interactions are
important. These involve non-covalent bonds such as hydrogen bonds
and ionic bonds, van der Waals attractions, and hydrophobic
interactions where the hydrophobic parts of R-groups tend to associate
together.
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Primary (1°) structure
The primary structure of a protein is the sequence of amino acid
residues in the polypeptide chain. The chain of amino acids has an
amino terminus at one end and a carboxyl terminus at the other,
reflecting the structure of the amino acid molecules. In writing out
primary structures of proteins, the convention is to write from the
amino terminus (N-terminus) on the left to the carboxyl terminus (Cterminus) on the right. In some cases the structure of the amino acid
residues can be shown, although mostly the abbreviated names for the
amino acids (either the three-letter abbreviations or the single-letter
designations) are used. Representations of primary structure are shown
in Fig. 2.3.4.
Fig. 2.3.4: Primary structure of a polypeptide. This could be written out
in full, but is usually abbreviated to save space. In (a) a diagrammatic
representation of the polypeptide is shown, with the N- and C-termini
labelled. (b) shows the three-letter abbreviations for a short peptide
sequence, and (c) shows the single-letter abbreviations for the same
sequence. The N- and C-terminus labels may be omitted; if so the
convention is that the N-terminus is on the left.
Secondary (2°) structure
There are two main types of secondary structure. These are the α -helix
and the β -sheet arrangements, which are generated from interactions
between the atoms of the amino acid residues in the polypeptide chain.
In the α-helix, as shown in Fig. 2.3.5, the polypeptide chain is coiled
into a right-handed helix by hydrogen bonds between the N-H group of
a peptide bond and the C=O group of the peptide bond four residues
away from it.
In the β-sheet configuration, polypeptide chains are linked together in a
side-by-side configuration, again by hydrogen bonding. Beta sheets can
be either parallel or antiparallel, depending on the orientation of the
constituent parts of the arrangement. The two types of β-sheet are
shown in Fig. 2.3.6.
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Fig. 2.3.5: The α-helix. This secondary structure is a right-handed helix
with four residues per turn. Hydrogen bonds between oxygen and
hydrogen atoms stabilise the helix. Not all H-bonds are shown.
Fig. 2.3.6: The β-sheet arrangement of secondary structure can be either
parallel or antiparallel. In the parallel arrangement the polypeptide
chains run in the same direction with respect to their N/C polarity. In
the antiparallel arrangement, the chains run in opposing directions.
Hydrogen bonds are shown by dotted lines.
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Tertiary (3°) structure
The α-helix and β-sheet arrangements are relatively simple ways to
organise stretches of polypeptide chain. More complex structures are
found when we look at the tertiary structure of proteins, which
describes the way in which the polypeptide folds to give the final
protein structure. Given that a polypeptide may be several hundred
amino acid residues in length, and that both α-helix and β-sheet
arrangements may be found in the same protein, it is easy to see that
predicting and analysing protein structure is a difficult task. Modern
computer graphics programmes enable scientists to look at protein
structure in ways that were not possible a few years ago, and many new
insights have come from computer modelling coupled with
experimental techniques such as examining protein crystals by X-ray
crystallography and nuclear magnetic resonance (NMR).
The tertiary structure of a protein is determined largely by hydrophobic
interactions, which place the non-polar R-groups in the centre of the
molecule. In many proteins, an additional important type of covalent
bond is the disulphide bond, which can form between the sulphydryl
(SH) groups on cysteine residues in different parts of the polypeptide
(or between cysteine residued in two different polypeptides). Within any
tertiary structure, parts of the amino acid sequence may adopt an αhelix arrangement, others may be β-sheets or more complex
arrangements of the β-sheet structure. A representation of the tertiary
structure of myoglobin is shown in Fig. 2.3.7.
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Fig. 2.3.7: Tertiary structure of myoglobin. This is a single polypeptide
chain with several α-helices linked by non-helical regions. Folding of the
protein produces a hydrophobic ‘pocket’ for the haeme group, which
contains the iron atom involved in binding oxygen. This representation
is a ribbon plot generated by the widely used RasMol computer software
package (copyright Roger Sayle, public domain). This programme
enables the structure to be viewed in many different ways such as balland-stick or spacefilled, and also permits rotation of the structure to
enable viewing from different angles. Software such as this is of great
benefit to protein chemists.
Folded proteins may have other non-protein groups associated with
them. These are called prosthetic groups, and are often essential for
the biological activity of the protein. Examples include the ironcontaining haeme group of myoglobin (see Fig. 2.3.7) and haemoglobin.
Proteins may also have carbohydrate, lipid or nucleic acid groups
associated with them; these conjugated proteins are known as
glycoproteins, lipoproteins and nucleoproteins, respectively.
The fact that proteins are relatively stable structures in the cellular
environment is remarkable in that the weak forces that hold the
structure together can be disrupted easily if the chemical environment
changes, or if the sequence of the amino acids is altered. Proteins fold to
give a structure with the lowest free energy, therefore each polypeptide
chain will have its own preferred conformation. Thus, although in
theory there are essentially unlimited possible protein structures, only a
small number of these possibilities will fold to give a single stable
conformation, which has enabled the evolution of protein structures
that are stable and uniquely suited for a particular purpose.
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Quaternary (4°) structure
Many proteins are made up of a single polypeptide chain, which folds to
a particular tertiary structure unique to that protein. Other proteins are
composed of two or more polypeptide subunits, each of which has its
own specific conformation. The organisation of the subunits in a multisubunit protein is known as the quaternary structure of the protein. A
good example is the tetrameric protein haemoglobin, composed of two
α- and two β-globin subunits, each with its own prosthetic haeme group.
Protein motifs and domains
As our knowledge of protein structure has improved, two additional
elements have been recognised within the traditional 1°/2°/3°/4°
classification. These are motifs and domains. Motifs are elements of
secondary structure that form in particular ways. Examples include the
β -α
α -β
β motif, which gives a fold or crease in the protein, and the
β -barrel, which forms a tube-like arrangement within the protein.
Domains are regions of the polypeptide chain that fold independently to
give structurally distinct regions of tertiary structure, often with
different roles to play in the complete protein.
Function of proteins
As might be expected from their structural complexity and diversity,
proteins have an equally wide range of different roles in the cell. One
common way of describing proteins is to group them as either fibrous
(structural) proteins, or globular (functional) proteins. However, this is
a rather simplistic classification, and it is better to classify proteins using
a more specific system that takes into account the many different and
highly specific roles that they carry out. Thus we have proteins that act
as enzymes, those that are found as structural components of cells and
tissues, receptor and signalling proteins, and many others. Some
examples of the types of function that proteins carry out in the cell are
shown in Table 2.3.8.
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Table 2.3.8: Some classes and functions of proteins in the cell.
Class of
protein
Function
Examples
Enzyme
Catalysis (breaking &
forming of covalent
bonds).
Thousands of examples! Usually
enzyme names end in -ase. Groups
include proteases, lipases,
polymerases, kinases, phosphatases,
isomerases, dehydrogenases, etc.
Structural
Provide support to cells
and tissues.
Collagen, elastin, tubulin, keratin,
actin.
Receptor
Detection and
transmission of signals.
Insulin receptor, acetylcholine
receptor, rhodopsin.
Signal
Intercellular signalling.
Insulin, other hormones & growth
factors.
Transport
Carries small molecules
or ions in bloodstream
or in membranes.
In the bloodstream, haemoglobin
carries oxygen, serum albumin carries
lipids, transferrin carries iron. Many
transmembrane proteins act as pumps
for transporting small molecules or
ions (protons, calcium, glucose)
across the membrane.
Motor
Generates movement.
Myosin in muscle is the most obvious
example, also dynein in cilia and
flagella. Kinesin interacts with
microtubules to move organelles.
Storage
Stores small molecules
or ions.
Ferretin stores iron in the liver.
Ovalbumin (egg white) and casein
(milk) are storage proteins.
Regulatory
Binds to DNA to regulate
gene activity.
The lac repressor binding to the
operator of the lac operon to switch
it ‘off’.
Special
purpose
Varied range of specialist
functions.
Glue proteins to attach mussels to
rocks, antifreeze proteins of Arctic
and Antarctic fish, plus many other
specialised examples.
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2.4 Nucleic acids
The information-carrying molecules of the cell are the nucleic acids DNA
(deoxyribonucleic acid) and RNA (ribonucleic acid). When compared
to the complexity of proteins, nucleic acids have a relatively simple
chemical structure that is based on a sugar-phosphate backbone. The
variable information-coding part of nucleic acids is made up of a set of
four nitrogenous bases, which arrange themselves in pairs. One of the
most fascinating aspects of molecular biology is how this very simple
coding system enables the great diversity of protein molecules to be
constructed. As we shall see, the answer is yet again another elegant
example of one of our main themes – that of emergent properties.
Nucleotide structure
The monomeric units of nucleic acids are the nucleotides. These are
composed of a pentose sugar (ribose in RNA, deoxyribose in DNA)
and a phosphate (PO 4) group that make up the constant structural part
of the molecule, and a variable nitrogenous base. The bases are either
purines (double-ring structures) or pyrimidines (single-ring
structures). This difference has important consequences for the way in
which the bases join together in the DNA molecule. The purine bases
are adenine and guanine (A and G, found in DNA and RNA). The
pyrimidines are cytosine (C, DNA and RNA), thymine (T, DNA only)
and uracil (U, found in RNA instead of thymine). Nucleotide structure is
shown in Fig. 2.4.1.
Fig. 2.4.1: Nucleotide structure. This is based around a 5-carbon sugar
(shaded) with a phosphate and a nitrogenous base attached to the C5
and C1 positions respectively. In RNA the sugar is ribose, with an OH
group at position X on C2. In DNA the sugar is deoxyribose, with X being
a hydrogen.
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The phosphodiester bond
As with the other macromolecules, a dehydration synthesis reaction
generates the bond that joins two nucleotides together. This
phosphodiester bond is formed between the phosphate group of one
nucleotide and a hydroxyl group on the sugar of the next nucleotide, as
shown in Fig. 2.4.2.
Fig. 2.4.2: Nucleotides are joined together by a phosphodiester linkage
between the C5 phosphate and the hydroxyl group on C3 (shaded area).
This gives polynucleotides a defined polarity reflecting that of the
component nucleotides.
Polynucleotides and nucleic acid function
Polynucleotide chains provide the structural and functional basis for the
encoding and decoding of genetic information. The sugar/phosphate
‘backbone’ of a polynucleotide carries the sequence of bases that makes
up the genetic code as a series of triplet codons. The functional basis of
the code is that of base-base recognition. Bases can fit together and join
by hydrogen bonding, A with T (or U) and G with C, as shown in Fig.
2.4.3. Bases that fit together in this way are said to be complementary.
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Fig. 2.4.3: Base pairing between purines and pyrimidines in the DNA
double helix. To fit the helix, one purine (A or G) must pair with one
pyrimidine (T or C). This gives a stable arrangement of A:T and G:C base
pairs, with two hydrogen bonds in an A:T bp and three in a G:C bp. Not
all the atoms are shown or labelled in this representation.
Whilst it may be rather too simplistic to say that one aspect of biology is
more important than any other, without base pairing there could be no
storage, replication, encoding or decoding of the information needed to
make all the components and systems found in living organisms. Thus
the A:T and G:C base pairs found in DNA have a significance far greater
than that which their relatively simple chemistry would suggest.
The DNA molecule is a double-stranded helix, the structure of which
was proposed by James Watson and Francis Crick in 1953. This double
helix (not to be confused with the α-helix of proteins!) is undoubtedly
the biological structure that is most widely recognised by non-scientists.
As with all the molecules and macromolecules that we have considered,
it is an elegant example of the interdependence of structure and
function. The double helix has two polynucleotide chains that run in
different directions (known as an antiparallel arrangement). The bases
fit across the centre of the right-handed helix, with one purine pairing
with its complementary pyrimidine. The significance of the sizes of the
purines and pyrimidines can now be appreciated, as the helix can
accommodate only a purine:pyrimidine base pair if the hydrogen bonds
are to be stable. The double helical arrangement of DNA is shown in Fig.
2.4.4.
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Fig. 2.4.4: The double helix of DNA. Two polynucleotide chains are
twisted into a right-handed helix. The chains are linked together by A:T
and G:C base pairs. The chains are in an antiparallel arrangement
(polarity indicated by arrows). The pitch of the helix is 3.4nm, diameter
2nm. The arrangement produces grooves on the surface of the helix.
(Copyright D S T Nicholl/Cambridge University Press. Reproduced with
permission.)
The structure of RNA differs from that of DNA in a number of ways. The
sugar in RNA is ribose, and the base uracil replaces thymine. Also, RNA
molecules are generally single-stranded, as in messenger RNA (mRNA),
although they do fold to give the 3-D conformations seen in ribosomal
RNA (rRNA) and transfer RNA (tRNA).
Complementary base pairing holds the key to the copying of genetic
information in the processes of DNA replication and transcription,
which are carried out by polymerase enzymes. The replication of DNA,
where each DNA strand is used as a template for the synthesis of a
complementary polynucleotide, is a critical part of the cell cycle.
Replication enables a complete copy of the genome to be passed on to
each daughter cell during mitosis. Transcription of genetic information
(into RNA from a DNA template) provides the mechanism for the
expression of genes.
In addition to polymerases, many other types of enzyme are associated
with nucleic acid biochemistry, both in the cell and as part of the genetic
engineer’s ‘toolkit’ that makes it possible to manipulate DNA in the test
tube. One example is DNA ligase, which forms phosphodiester bonds
and can be used to join DNA molecules together to create recombinant
DNA (rDNA).
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2.5 Cell membranes
The cell membrane or plasma membrane is of fundamental importance,
as it represents the barrier that separates the cell contents from the
extracellular environment. Also, as we have already seen, eukaryotic
cells use membranes to generate compartments within the cell, each
with a specialised function. In discussing membranes we will concentrate
on the plasma membrane (PM), although many of the features of the PM
are shared by the other membranes in the cell.
We can recognise several important functions of membranes, as follows:
• Providing selectively permeable barriers – the PM prevents
unrestricted movement of materials by acting as a barrier. However, it
also enables substances to enter and leave the cell by being selectively
permeable, a feature that is also critical for internal membrane
function.
• Compartmentalisation – membranes are used extensively in
eukaryotic cells to form structures such as the nuclear envelope,
endoplasmic reticulum, Golgi apparatus, mitochondria and
chloroplasts.
• Localising reactions in the cell – membranes provide the structural
framework for organising many of the reactions in the cell as a
consequence of compartmentalisation. Also, critical energytransducing mechanisms such as the light reactions of photosynthesis
and the respiratory electron transport chain are closely associated
with membranes.
• Transport of solutes – in addition to their selective permeability,
membranes have the machinery necessary for transporting solutes
specifically across the membrane, often against a concentration
gradient.
• Signal transduction – receptors on the membrane surface recognise
and respond to different stimulating molecules, enabling specific
responses to be generated within the cell.
• Cell-cell recognition – the external surface of the membrane is
important in that it represents the cell’s biochemical ‘personality’. In
multicellular organisms this feature enables cells to recognise each
other as similar or different, which is necessary for the correct
association of cells during development.
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Membrane structure
The basic structural unit of membranes is the phospholipid bilayer, as
outlined in Fig. 2.2.4. The proposal that this was the basis of membrane
structure was put forward in 1925, following a simple calculation about
the surface area (SA) covered by the lipids extracted from red blood
cells. It was found that this was roughly twice that of the SA of the cell,
and it therefore seemed likely that the lipids formed a double layer
surrounding the cell.
The currently accepted model for membrane structure is based on that
of Singer and Nicolson, who proposed the fluid mosaic model in 1972.
In this structure the phospholipid bilayer has proteins embedded within
it (intrinsic or transmembrane proteins) or associated with its surface
(extrinsic or peripheral proteins). Other components include
cholesterol, glycoproteins and glycolipids. The membrane is not a static
or rigid structure, but is a dynamic arrangement of lipids and proteins.
Thus the components of membranes are able to drift laterally within the
membrane, and therefore the term fluid mosaic is an accurate
description of the nature of the membrane. The fluid mosaic model is
shown in Fig. 2.5.1.
Fig. 2.5.1: The fluid mosaic model of membrane structure. The
phospholipid bilayer contains partially- and fully-embedded proteins,
cholesterol, glycolipids and glycoproteins. The structure is a dynamic or
fluid structure, in which the proteins and other components are in a
state of continuous lateral drift or motion.
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Function of membrane proteins
Proteins make up around 50% of the mass of the PM, and can be
classified into different groups according to either their arrangement in
the membrane and/or their function. As shown in Fig. 2.5.1, proteins
may be embedded in the lipid bilayer or attached to the surface. The
embedded (intrinsic) proteins may be transmembrane proteins or they
may be linked to the lipids in one side of the bilayer only. The extrinsic
or peripheral proteins are loosely attached to the membrane by noncovalent association with other proteins.
When classifying membrane proteins according to function, several types
can be recognised. Transport proteins, as the name suggests, are
involved in transporting non-diffusible substances across the membrane.
Transport proteins may be either channel proteins or carrier proteins.
Channel proteins provide a ‘pore’ across the membrane, through which
substances (usually small charged molecules) can diffuse. Carrier
proteins are more specific, with binding sites for one type of solute only.
Both channel and carrier transport proteins can permit passive
transport (with the concentration gradient, sometimes called facilitated
diffusion). To transport molecules against the concentration gradient,
special types of carrier proteins are needed that can harness energy to
drive the transport process during active transport.
In addition to transport functions, membrane proteins are important as
enzymes and receptors. They have a role to play in cell adhesion and
cell-cell recognition (glycoproteins; glycolipids are also important) and
provide a structural support to the cell as part of the membrane-linked
cytoskeleton. Membrane protein function is shown in Fig. 2.5.2.
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Fig. 2.5.2: Function of membrane proteins. (a) Channel protein.
(b) Carrier protein, shown here as an active transport protein which
requires ATP hydrolysis to transport molecules against the
concentration gradient. (c) Membrane-bound enzyme, converting
substrate X to product Y. (d) Receptor protein, which generates an
intracellular response (R) to an extracellular signal (S).
(e) Cell adhesion protein, linking two cells together. (f) Cell:cell
recognition via a glycoprotein. (g) A membrane protein attached to the
cytoskeleton. Sometimes a single protein may perform more than one of
these functions.
2.6 The cytoskeleton
The eukaryotic cell has an intricate network of thread-like filaments
called the cytoskeleton, which supports the interior of the cell and the
organelles within it. The cytoskeleton is made up of three components.
In order of increasing diameter these are actin filaments (also known
as microfilaments), intermediate filaments and microtubules.
Microfilaments are composed of the protein actin, arranged as two
strands of protein molecules twisted together to give a rope-like
structure about 7nm in diameter. These are present throughout the cell,
but are most highly concentrated just inside the plasma membrane.
Microfilaments are important in cell movements.
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Intermediate filaments are about 10nm in diameter and are composed of
tough fibrous protein strands twisted together. This means that they are
very stable structures in the cell, and are particularly important for
providing mechanical strength to animal cells, which lack the strong cell
walls of plants. They may be anchored to the cell membrane to provide
support.
Microtubules are hollow tubes made up of the protein tubulin.
Heterodimers (one α- and one β-tubulin subunit per dimer) are
arranged as a set of 13 protofilaments to generate the final
microtubule, which is a relatively rigid structure. Microtubules are
important in cell division as part of the spindle fibre network, and are
also involved in the movement of components within the cell.
Microfilaments and microtubules can de-polymerise and re-polymerise
very easily. Thus the cytoskeleton is not a static structure, but a dynamic
one, which is continually changing to provide the basis of support and
movement in the cell. The control of microtubule polymerisation and
depolymerisation in animal cells is controlled by the centrosome or
microtubule organiser. In interphase, the centrosome serves as the
site for the production of cytoplasmic microtubules that make up part of
the cytoskeleton. During mitosis, the microtubules are re-deployed as
the spindle fibres that separate the chromatids to generate the daughter
nuclei.
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SECTION 3
In the previous sections we have looked at how cells are put together,
concentrating mainly on the structure of components such as molecules
and macromolecules, membranes and organelles. In this section we
probe a little deeper into some examples of how the cell actually works
as a molecular machine, by examining the interactions that make the
link between structure and function. We will consider three examples:
catalysis by enzymes, the sodium-potassium pump, and cell signalling.
3.1 Catalysis
Enzymes are the biological catalysts of the cell. They increase the rate at
which reactions happen, often by millions of times. Only by having this
set of very specific catalysts can the cell carry out all the reactions that
are necessary for metabolic processes to work effectively. Some of the
types of enzymes were listed in Table 2.3.8, and it is now useful to
describe a few of these in more detail.
Enzymes that break things apart include:
• hydrolases – these enzymes catalyse a hydrolytic cleavage reaction.
• phosphatases – remove a phosphate from a molecule by a hydrolytic
cleavage.
• proteases – hydrolyse peptide bonds to break down proteins into
amino acids.
• nucleases – hydrolyse phosphodiester bonds to break down nucleic
acids into nucleotides.
• ATPases – hydrolyse ATP. Many proteins have an ATPase activity which
is essential for their function.
Enzymes that join things together include:
• kinases – catalyse the transfer of a phosphate group onto a molecule
such as a carbohydrate or a protein.
• synthases – these enzymes join two molecules together by catalysing
a dehydration synthesis reaction.
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• polymerases – catalyse polymerisation reactions where molecules are
added sequentially to a chain, as in DNA and RNA synthesis.
Other types of enzyme are:
• oxidoreductases – catalyse electron transfer oxidation/reduction
reactions. Include the oxidases, reductases and dehydrogenases
(found in, for example, respiration and photosynthesis).
• isomerases – change the form of a molecule without altering its
overall chemical composition.
Most enzymes are now named according to their substrate and their
function, although a few are named inconsistently due to historical
reasons. Enzymes are also given a standard reference number (the EC
number) to help characterise the 1,500 or so that are now known in
detail. Examples of enzyme names are glucose oxidase, glyceraldehyde
3-phosphate dehydrogenase, citrate synthase, phosphofructokinase,
and (the most important one on earth!) ribulose bis-phosphate
carboxylase.
Form and function
Enzymes work by bringing the substrate(s) of a reaction close together
in the active site region so that bond breakage or formation may occur
at the atomic level. This is often facilitated by specific chemical effects
such as the transfer of protons or the alteration of charge distribution
around the target atoms. As might be expected, the substrate and
enzyme must fit together very precisely. We can see this most easily by
looking at an enzyme that has a large substrate (i.e. of a similar size to
the enzyme itself). This is shown in Fig. 3.1.1 – in this case, a restriction
endonuclease and its substrate, which is a defined sequence in the DNA
molecule.
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Fig. 3.1.1: The interaction of the enzyme BamHI (a restriction
endonuclease) with its DNA substrate. The enzyme is shown as a RasMol
ribbon plot with the DNA helix in black. In (a) the view is from the side,
in (b) it is along the axis of the DNA helix. The way in which the enzyme
wraps around the DNA is shown clearly.
The catalytic cycle
As enzymes are catalysts, they remain unchanged at the end of a
reaction, and are available for the next interaction with substrate. This
catalytic cycle can be represented as shown in Fig. 3.1.2. This shows
the enzyme sucrase, which catalyses the hydrolysis of sucrose into its
component monosaccharides, glucose and fructose. At the start of the
cycle, enzyme (E) and substrate (S) are available. The molecular
interaction of enzyme and substrate at the active site forms the
enzyme:substrate (ES) complex. Catalysis occurs, forming the
enzyme:product complex (not shown in the diagram) and the products
are released, which frees the enzyme for the next substrate molecule.
Fig. 3.1.2: The catalytic cycle of sucrase. The enzyme hydrolyses the
disaccharide sucrose, releasing glucose (G) and fructose (F). The
enzyme remains unchanged at the end of the reaction cycle.
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A common model for enzyme action is the lock and key model, which is
often represented as shown in Fig. 3.1.2. However, this model is a little
misleading in that it tends to give the impression that enzymes are rigid
structures, whereas in fact they are quite flexible and can alter their
conformation in response to the binding of other molecules. The
currently accepted model for enzyme action is the induced fit model, in
which conformational changes to the protein occur on binding of the
substrate. We will look at the action of the enzyme hexokinase to
illustrate the induced fit model.
Hexokinase catalyses the transfer of a phosphate from ATP onto glucose.
The structure of the enzyme is shown in Fig. 3.1.3. The active site and
the two domains of the single polypeptide chain are clearly visible in
this view of the backbone of the molecule. You might think of the
protein about to close round the substrate in the active site in a similar
way to your hand closing round a door handle. The effect of this is that
glucose fits the active site more closely, and the binding of ATP is also
enhanced. The catalytic cycle of hexokinase is shown in Fig. 3.1.4. It may
help to think of the protein ‘flexing’ during the binding of substrate.
Fig. 3.1.3: The structure of hexokinase. This is a RasMol plot of the αcarbon/peptide bond backbone (repeated C-C-N-C sequences) of the
polypeptide. The protein is a single polypeptide chain, with two distinct
domains that create the pocket where the active site is found. The
domains are shown by the dotted line.
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Fig. 3.1.4: The catalytic cycle of hexokinase. (a) Enzyme and substrate
are apart. (b) Substrate binds to active site region. (c) A conformational
change in the protein closes the active site around the substrate
(induced fit). Binding of ATP is enhanced, catalysis occurs and the
phosphate is transferred to glucose. (d) Enzyme plus products.
Control of enzyme activity
The activity of enzymes must be regulated in some way if metabolic
chaos is to be avoided. Regulation can be achieved by a number of
different mechanisms. A major influence is the number of enzyme
molecules in the cell, which is controlled at the level of gene
expression. Compartmentalisation also enables the cell to keep sets of
enzymes together and away from other enzymes, and temperature and
pH also affect enzyme activity. Many enzymes also require cofactors to
function. However, the most effective way of enabling a fine control of
enzyme activity is to alter the shape of the enzyme itself, and thus cause
a change in its catalytic efficiency. Examples of this type of metabolic
control include inhibitors, allosteric effects, covalent modification,
and end-product inhibition.
Inhibitors can be either competitive or non-competitive. As the name
suggests, competitive inhibitors compete for the active site, thus
reducing the effectiveness of the enzyme. Competitive inhibitors are
usually similar in structure to the substrate, and the enzyme is ‘fooled’
into accepting the inhibitor, which blocks the active site. Noncompetitive inhibitors bind at a different location and change the
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conformation of the protein, altering the active site and again reducing
catalytic efficiency. Inhibition can be either reversible or nonreversible, depending on how the inhibitor binds to the enzyme. A
diagram of enzyme inhibition is shown in Fig. 3.1.5.
Fig. 3.1.5: Enzyme inhibition. (a) shows competitive inhibition, with the
inhibitor occupying the active site. In (b) non-competitive inhibition is
shown, with the inhibitor binding to a site that is separate from the
active site region.
Allosteric enzymes are those that ‘change form’ in response to binding
of a regulating molecule (often called a modulator or effector).
Allosteric modulators can be either positive or negative effectors of
enzyme activity. They function by binding to allosteric sites that are
distinct from the active site of the enzyme. As shown in Fig. 3.1.5, noncompetitive inhibition is a form of allosteric regulation. In multi-subunit
enzymes, the structure is more complex, and the enzyme often exists in
two different conformational states (active and inactive). These can be
stabilised by binding the modulator; positive modulators stabilise the
active form of the enzyme, whilst negative modulators stabilise the
inactive form. This is shown in Fig. 3.1.6. In addition to positive and
negative modulators changing the activity of an allosteric enzyme,
sometimes binding of the substrate itself to one active site enhances
binding at the other active sites. This is known as cooperativity.
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Fig. 3.1.6: Allosteric effects shown for an enzyme with four subunits.
There is an equilibrium between active and inactive forms of the enzyme
(a), with the active form able to bind the substrate. In (b) the effect of
binding a positive modulator is shown. This binds to an allosteric site
and stabilises the active form of the enzyme. The effect of a negative
modulator is shown in (c); in this case the inactive form is stabilised.
Covalent modification of enzymes is another strategy that is used
widely in metabolic regulation. One of the most common additions is a
phosphate group, which can alter the shape of a protein by attracting
positively charged R-groups (as phosphates carry two negative charges
on the two single-bonded oxygen atoms). Protein kinases add
phosphate groups, and phosphatases remove them, thus the effect can
be reversed. Some proteins are activated by phosphorylation, others are
inactivated (Fig. 3.1.7).
Fig. 3.1.7: General outline of protein phosphorylation effects. Kinase
and phosphatase enzymes add or remove phosphates from enzymes. In
(a) the effect of phosphorylation is to increase activity (shown by the +
sign). In (b) the effect is to inactivate the enzyme (– sign).
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An example of phosphorylation activating an enzyme is the skeletal
muscle enzyme glycogen phosphorylase. This is used to generate
glucose from glycogen when heavy demands are placed on muscle
tissue. The enzyme is present as an inactive non-phosphorylated form,
which is converted to the active form by the addition of a phosphate to a
serine residue in the protein by phosphorylase kinase. When energy
demand falls off, phosphorylase phosphatase removes the phosphate
group and inactivates the enzyme. However, this is not the whole story
– glycogen phosphorylase is also regulated by an allosteric effect.
Glucose and ATP act as negative modulators, and AMP acts as a positive
modulator. This avoids having full activation by the phosphorylation
alone, and provides a regulatory mechanism that is responsive to the
ATP/AMP ratio in the cell. A further complication is that there is a
hormonal control mechanism by epinephrine and glucagon. This
illustrates very clearly the complexity of metabolic regulation, with often
two or more distinct mechanisms used to control enzyme activity in a
precise and responsive way.
Yet another form of control by a covalent activating mechanism is
proteolytic cleavage, as found in the enzyme trypsin (a protease,
important in digestion). It is synthesised in the pancreas, but must not
be made in its active form, or it would digest the pancreatic tissue itself!
Thus it is synthesised as a slightly longer protein called trypsinogen),
which is inactive (the general name zymogen is used to describe these
inactive protease precursors). Activation occurs when trypsinogen is
cleaved by a protease in the duodenum. Once active, trypsin can activate
more trypsinogen molecules, resulting in an autocatalytic cascade that
produces a large number of active trypsin molecules very rapidly.
Trypsin also cleaves other zymogens such as chymotrypsinogen.
Cascades like this are another important part of the overall metabolic
regulatory strategy of the cell.
So far we have built up a picture of the complex mechanisms used to
control the activity of specific enzymes. However, metabolism is
organised as a series of metabolic pathways, and control of these
pathways is an important feature of cell biochemistry. One way in which
control can be exercised is by end-product inhibition, as shown in Fig.
3.1.8. Regulating the output of a biochemical pathway according to the
amount of its product is an efficient mechanism, as it avoids the wasteful
conversion of the intermediates in the pathway. This is a form of
negative feedback.
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Fig. 3.1.8: End-product inhibition in a metabolic pathway. A, B, C and D
are the substrates/products of the enzymes (1, 2 and 3). When D
increases in concentration, it can bind to the first enzyme in the
pathway and reduce the efficiency of conversion of A to B. This in turn
controls the whole pathway as the supply of intermediates is restricted.
3.2 The sodium-potassium pump
The movement of solutes against a concentration gradient by active
transport is an essential part of the cell’s metabolism. One of the best
examples of how this works is the sodium-potassium pump (Na+/K+
pump) in animal cells. This transports sodium ions out of cells, and
potassium ions in. The pump is driven by the hydrolysis of ATP. The
overall result is that the intracellular concentration of Na+ is kept low
compared to the external concentration. For K+ the opposite applies –
the intracellular concentration is high compared to the outside.
Maintaining this imbalance of intracellular/extracellular Na+ and K+
concentrations is one of the cell’s most critical functions, and it accounts
for around 30% of the energy expenditure of a typical animal cell. A
summary of the features of the Na+/K + pump is shown in Fig. 3.2.1.
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Fig. 3.2.1: Summary of the sodium-potassium pump. The membranebound protein complex transports sodium ions to the extracellular
environment, and potassium ions to the intracellular environment. The
pump is driven by ATP-derived phosphorylation, which induces
conformational changes in the protein. Three sodium and two
potassium ions are exchanged for each cycle of operation. This results in
the maintenance of Na+/K + concentration (electrochemical) gradients as
shown.
The pump is a very elegant example of our theme of structure and
function being closely related. The protein complex is actually made up
of four subunits; two large and two small. We will consider the protein
complex as a single unit for simplicity. The key features of the pump are:
• it is a transmembrane carrier protein
• it has three binding sites for Na+ ions
• it has two binding sites for K + ions
• there is a phosphorylation site to accept a phosphate from ATP
• two different conformations of the protein are possible (controlled by
the phosphorylation state).
As the protein works by phosphorylation, and ATP is the source of the
phosphate group, the whole complex is often called the
Na+/K+– ATPase. The overall pattern is that the hydrolysis of one ATP
molecule drives the export of 3 sodium ions and the import of 2
potassium ions.
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The way in which the pump works is shown in Fig. 3.2.2. Initially, the
pump is open to the cytosol. Sodium ions can bind to the Na+ binding
sites, and the ATPase function phosphorylates the protein. This causes a
conformational change, which makes the protein open to the
extracellular environment. The Na+ binding sites now have a lowered
affinity for sodium, and the Na+ ions are released. The K+ binding sites
(high affinity for K+) can then be occupied; again, a conformational
change occurs, with the release of the phosphate, which switches the
protein back to the ‘open to inside’ structure. The K+ sites now have a
lowered affinity for potassium, and the K+ ions are released. This cycle is
repeated, with as many as 300 sodium ions being transported per
second.
Fig. 3.2.2: The mechanism of action of the sodium-potassium pump. The
membrane is shaded, with the external environment at the top of each
diagram. Sodium ions are black squares, potassium ions are black
circles. (a) The protein is in the ‘open to cytoplasm’ conformation,
which allows sodium ions to bind. (b) Phosphorylation of the protein.
(c) A conformational change releases sodium to the external
environment. (d) Potassium binds to the binding sites now exposed to
the external environment. (e) Dephosphorylation of the protein restores
the ‘open to inside’ conformation. (f) Potassium ions released; the pump
is now ready for the next cycle of operation.
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The Na +/K+ pump is an excellent example of a ‘molecular machine’, in
which two features work in concert. Firstly, the conformational change
in protein structure caused by ion binding and phosphorylation/
dephosphorylation provides the mechanism of action of the pump.
Secondly, the alteration of the affinity for Na + and K+ ion binding
enables the pump to capture and release the ions at the correct points
in the pumping cycle.
3.3 Cell signalling
Although cells are in many ways self-contained units, they do not exist in
isolation. Even a unicellular organism must detect and respond to
outside influences – chemicals, light, nutrient availability, and the
presence of other cells. In multicellular organisms, the organisation of
cells into tissues and systems brings added complexity. It is therefore
essential that cells are able to ‘talk’ to each other by a set of processes
that can be grouped under the general heading of cell-cell
communication or cell signalling. In this section we will consider how
this is achieved in animal cells.
General principles
Communication between cells is very much like any other form of
communication – it involves transmitting and receiving information. A
signal is sent by a signalling cell and received by a target cell. Where a
change in the form of the signal is required, this is signal transduction
(faxing a letter involves signal transduction – conversion of the printed
form into the electronic form). Signal reception and transduction are
the two areas of cell signalling that most is known about.
Communication can be achieved by a number of different systems:
• endocrine – secretion of a hormone into the bloodstream, which
enables dispersal throughout the body. The signalling cell and the
target cell can be far apart.
• paracrine – works over a more restricted area than endocrine
signalling. A local mediator is secreted, which can affect cells in the
immediate area of the signalling cell.
• neuronal – nerve cells or neurones elicit responses by the release of
a neurotransmitter at synapses. Like hormones, the signal can cover
long distances; in this case, through the network of nerve cells rather
than in the bloodstream.
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• contact-dependent – signal molecules in the plasma membrane of the
signal cell interact with membrane-bound receptors on the target cell.
These signals are therefore restricted to cells that are in direct
contact.
Often similar types of molecules are used in these different forms of
signalling. Examples include amino acid derivatives such as adrenaline
(hormone), histamine (local mediator) and GABA (γ-aminobutyric acid,
an inhibitory neurotransmitter). Many signal molecules are proteins,
such as insulin (hormone) and EGF (epidermal growth factor, a local
mediator). At the other end of the scale in terms of complexity is the
local mediator nitric oxide (NO), which causes relaxation of smooth
muscle cells in blood vessels, dilation and therefore increased blood
flow. Another important group of signalling molecules are the steroid
hormones, which are based on the cholesterol four-ring structure (as
outlined in Fig. 2.2.5).
In multicellular organisms, there may be hundreds of different signal
molecules. Responses to these can be controlled by variations in the
reception at the cell surface. In some cases, one signal/receptor is
required, whilst in others multiple signals are involved. The signal can
also be interpreted in different ways within the cell, often by a cascade
system involving modulation of the signal, amplification, and the
generation of different responses by different parts of the cell. Thus the
signalling system can be controlled in a very precise way; this is
obviously critical if the correct response is to be generated.
Extracellular hydrophobic signalling molecules
Some small hydrophobic molecules can cross the plasma membrane and
enter the cell by diffusion. The best known classes are the steroid
hormones (such as cortisol and testosterone) and the thyroid
hormones (such as thyroxine; see Fig. 3.3.1). They work by activating
gene regulatory proteins in the cell, which stimulate transcription of
particular sets of genes in the nucleus. The hormones can diffuse across
the plasma membrane and bind to receptor proteins that are located
either in the cytosol or in the nucleus itself. The mode of action of
cortisol is shown in Fig. 3.3.2.
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Fig. 3.3.1: Structure of the thyroid hormone thyroxine. This is an amino
acid derivative with two aromatic rings and four iodine atoms.
Fig. 3.3.2: Cortisol works by diffusing across the plasma membrane and
binding to an intracellular receptor protein (RP). This enters the nucleus
and binds to a regulatory site on the DNA, stimulating transcription.
Extracellular hydrophilic signalling molecules
In contrast to the hydrophobic signals outlined above, the majority of
signalling molecules are either too large or too hydrophilic to cross the
plasma membrane. The receptor proteins for these signals must
therefore present a binding site to the extracellular environment, and
elicit a response in the cytosol. There are three main classes of these cell
surface transmembrane receptors:
• ion-channel-linked
• enzyme-linked
• G-protein-linked
These types of receptor all bind extracellular signal molecules, but
generate intracellular responses in different ways. A summary of their
mode of action is shown in Fig. 3.3.3.
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Fig. 3.3.3: The three classes of receptor proteins. (a) Ion-channel-linked
receptors. In this diagram the channel is ‘closed’ and ions cannot pass
through. Binding of the signal (S) causes a conformational change,
‘opening’ the channel. (b) Enzyme-linked receptors, with the inactive
enzyme converted to the active form on binding of the signal molecule.
(c) G-protein-linked receptors (R) activate the G-protein (G) in response
to signal binding. The G-protein acts on a target protein (T), which may
be an enzyme or an ion-channel protein. This generates the intracellular
response.
Ion-channel-linked receptors (also known as chemically-gated ion
channels) open pores through the protein in response to binding of
the signal molecule. Ions flow through this ‘gate’, generating an
electrical effect. This type of receptor is found in excitable cells such as
nerve cells and muscle cells. Enzyme-linked and G-protein-linked
receptors are found in all types of cells. The enzyme-linked receptors
generate an enzyme activity (usually a kinase activity) on the cytoplasmic
end of the protein. This kinase activity causes the phosphorylation of
other intracellular proteins, thus activating them. G-protein-linked
receptors activate a GTP-binding protein (the G-protein) that sets off a
chain of events in the cell. This group of receptors is the largest known,
and many different signals and responses can be associated with Gprotein activity. Let’s look at how G-protein-linked receptors work in a
little more detail.
Although many different variations exist, all G-protein-linked receptors
have the same structural arrangement within the membrane. This is
known as a seven-pass transmembrane protein, as shown in Fig. 3.3.4.
Several hundred types of receptor are known, which bind signals as
diverse as peptide hormones, amino acids, fatty acids, and
neurotransmitters. On binding the signal, the G-protein is activated by
binding of GTP. The activated protein diffuses away from the receptor
protein site, and activates its target protein. This may be an ion-channel
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protein, or an enzyme such as adenylate cyclase or phospholipase C.
These enzymes catalyse the formation of small molecules known as
second messengers, which trigger the intracellular response to the
original signal transduction event at the cell surface. Adenylate cyclase
activity generates cyclic AMP (cAMP), phospholipase C generates
inositol triphosphate (IP3). Second messengers are important parts of
the signal transduction pathway, and can have many different effects. An
outline of the cAMP pathway is shown in Fig. 3.3.5.
Fig. 3.3.4: Structure of the G-protein-linked receptor family. The sevenpass transmembrane protein crosses the membrane seven times, and has
distinct binding sites for the extracellular signal molecule and the
intracellular G-protein.
Fig. 3.3.5: The cyclic AMP (cAMP) signal transduction pathway. The Gprotein-linked receptor binds the signal molecule and this causes
activation of the G-protein (GDP is exchanged for GTP). The activated
G-protein diffuses to its site of action – in this case, adenylate cyclase
(AC). This catalyses the formation of cAMP from ATP. The cAMP acts as a
second messenger, triggering a variety of intracellular reactions that
often form a cascade effect.
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Signal transduction is a complex topic. We have seen that signals can be
of many different types, and can act either by diffusing across the plasma
membrane (steroid hormones and nitric oxide) or by interacting with a
receptor protein on the cell surface. The variety of signals, receptors
and responses means that the system of signal reception and
transduction can generate very specific effects in different types of cell.
The response of a cell to a signal can involve ion flow, activation of
specific proteins, or changes in gene expression. These effects can be
short-lived, as in the case of the generation of an action potential, or
they may be permanent alterations that control the developmental fate
of the cell. It is therefore clear that the idea of a cell as a self-contained
unit is in fact very far from the reality of the situation – cells are
constantly engaged in the exchange of information in the form of
molecular signals, and it is this that enables cells in multicellular systems
to function in an integrated way.
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SECTION 4
One of the defining features of modern biology is the extensive use of
the technology of gene manipulation. The first recombinant DNA
molecules were constructed at Stanford University in 1972, and
developments since then have been truly staggering. It is now possible
to isolate genes relatively easily, determine their DNA sequence, and
assess their function. Genes can be modified in the test tube and
replaced in the original organism or in a different host, and many types
of transgenic plants and animals have been developed.
In this final part of our look at cell and molecular biology, we will
consider four of the areas in which gene manipulation technology is
having (or will have) a major effect on our lives – the human genome
project, human therapeutics, forensic science, and agriculture. Although
we will concentrate on these aspects, it is useful to remind ourselves
that the great advances in recombinant DNA technology all depend on
the simple rules of A:T and G:C base pairing. This is illustrated in Fig.
4.1, which shows the use of a radioactive probe in the identification of
DNA fragments in two common applications.
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Fig 4.1: Detecting DNA sequences by hybridisation. In (a) a radioactive
probe sequence (asterisks) is shown hybridising to its complementary
sequence. This can be used to detect cloned sequences in bacterial
colonies, as shown in (b). Clones with the desired sequence are
identified by the positive response on X-ray film, caused by the probe
binding to the complementary sequence on the filter replica of the
bacterial colonies. In (c) a similar process is shown for DNA fragments
in an electrophoresis gel. The fragments are transferred to a filter and
the probe sequence binds to complementary DNA sequences. The pattern
can be used to determine which fragments contain the desired sequence.
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Recombinant DNA technology is now part of our culture, for both the
scientist and for members of the public. Public concern about the use of
gene technology is an area that poses perhaps the greatest challenge to
the biological community over the next few years. If we are to gain from
the benefits that gene technology offers, and avoid the pitfalls, we will all
have to take an active part in the debates that lie ahead.
4.1 The human genome project
The genome of an organism is its complete complement of genetic
information. Most organisms have a DNA genome, although a few viruses
have RNA as their genetic material. In animal cells, the nuclear genome
is the major store of genetic information, with mitochondria having
their own smaller genome (mitochondrial DNA or mtDNA). In plants,
the chloroplast also has its own genome (chloroplast DNA or cpDNA).
The big project in biology at the moment is the human genome project
(HGP). The aim of this major international effort is to determine the
complete DNA sequence of the human genome, with a projected
completion date of 2003. In parallel with the human genome effort,
many other organisms are being investigated, with some genome
projects (such as E. coli and the yeast Saccharomyces cerevisiae) having
already been completed.
The obvious question in genome sequencing is – why do it? The answer
is that it will help us to understand the very nature of life itself, by
studying the information that organisms require to function. This area is
sometimes called bioinformatics, and there are many similarities
between bioinformatics and the great technical advances that have been
made in computing. Just as it is impossible to understand fully how a
computer works without examining the software that drives it, genome
projects aim to decipher the ‘software’ that makes organisms what they
are. As well as technical difficulties, this raises ethical problems. Many
people are concerned that information about their individual genetic
makeup could be used to discriminate against them in areas such as
medical care and life assurance.
In humans, the amount of information in the haploid nuclear genome is
some 3.0 × 109 base pairs (3,000,000,000 or 3 billion!). The structure of
the genome is complex, and different classes of DNA can be recognised.
About 40% of the total is either highly or moderately repetitive
sequence DNA. Of the remaining 60%, which represents unique
sequence and low copy number sequence elements, only around 3% is
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the actual coding sequence that makes up the estimated 60,000 – 80,000
genes that are needed to make a human being. The extra DNA is found
between genes as spacer DNA, and also within gene sequences
themselves. These intervening sequences or introns mean that
eukaryotic genes are much larger than is necessary to code for the
proteins that they specify. The RNA copy of the gene produced by
transcription therefore has to be ‘edited’ by a process known as RNA
splicing or RNA processing. Whilst the exact role of introns is still not
clear, it seems likely that they are important in regulating the expression
of genes and in generating new protein molecules during the process of
evolution.
When we consider the 3 billion base pairs in the genome, it is difficult to
know what this means in real terms. One example that is often used is to
consider writing out the genome sequence. Fig. 4.1.1 shows a short
stretch of DNA sequence. If we were to write out the human genome on
a paper ribbon using this size of typeface, we would need around 5,000
kilometres to represent the information – about the distance from
Glasgow to New York! Imagine trying to make sense of this amount of
information, with the only variation being the order of the four bases A,
G, C and T, and you will begin to get some idea of the scale of the
problem...
Fig. 4.1.1: The DNA sequence. If the human genome was written out like
this, you would need about 5,000km of this ribbon to represent the
entire sequence.
Another analogy may help to set the HGP in context. If you wish to
travel from Inverness to Plymouth, it is useful to follow a map. You
would not usually buy street maps of all the towns and villages between
Inverness and Plymouth, although in theory you could find your way by
doing this and linking all the hundreds of maps together. A more
sensible way would be to look at a road atlas of Britain, and see what
major destinations you would aim for – say Inverness, Glasgow, Carlisle,
Manchester, Birmingham, Bristol, Plymouth. You would then fill in more
detail as required – Inverness, Aviemore, Perth, Stirling, Glasgow, and so
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on. Your route would take you on particular roads – the A9, M74, M6
etc., so you could keep track of the destinations and the routes between
them.
Sequencing the genome is a bit like constructing a road map. The first
requirement is to pinpoint the major landmarks, keeping the
information in order, and then completing the picture with the detailed
sequence information. In the HGP this is achieved by three approaches:
• genetic mapping
• physical mapping
• DNA sequencing.
One particular problem is the interface between the techniques –
particularly the link between genetic and physical maps, which are based
on different criteria. Put simply, it is important to get the stretches of
sequence information in the right order! Let’s now consider how these
techniques contribute to the HGP.
Genetic linkage mapping
Linkage mapping can be used to locate genes on particular
chromosomes, and establish the order of these genes and the
approximate distances between them by determining their
recombination frequency. This approach relies on having genetic
markers that are detectable – sometimes these are genes that cause
disease, traced in families by pedigree analysis. The marker alleles must
be heterozygous so that meiotic recombination can be detected. If two
genes are on different chromosomes, they are unlinked and will assort
independently during meiosis. However, genes on the same
chromosome are physically linked together, and a crossover between
them during prophase I of meiosis can generate non-parental
genotypes. The chance of this happening depends on how far apart they
are – if they are very close together, it is very unlikely that a crossover
will occur between them. If far apart, they may behave as though they
are essentially unlinked. By working out the recombination frequency it
is therefore possible to produce a map of the relative locations of the
marker genes. A summary of recombination possibilities is shown in
Fig. 4.1.2.
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Fig. 4.1.2: Genetic linkage. In (a) two maternal (M) and two paternal
(P) chromosomes are shown (chromatids are not shown in this
diagram). Gene A (alleles A & a) and B (alleles B & b) are on different
chromosomes, and so will behave independently during meiosis. In (b)
the two genes D and E are linked; the paternal chromosome has D and e
alleles, the maternal has d and E. If a crossover (X) between non-sister
chromatids occurs between the loci for D and E, recombination can
generate non-parental genotypes in the gametes. The frequency of this
event occurring between loci can be used as a measure of how far apart
they are.
The overall result of genetic mapping is to produce a picture of the
locations of the marker loci on the chromosome – a bit like establishing
the order of the cities and large towns on the route from Inverness to
Plymouth, but not yet knowing the precise distances between them, or
the road numbers. Physical mapping is required to add some more of
the detail.
Physical mapping
Until the early 1980s it was thought that a physical map of the human
genome was unlikely to be achieved. As with genetic maps, construction
of a physical map requires markers that can be mapped to a precise
location on the DNA sequence. Physical maps of the genome can be
constructed in a number of ways, all of which have the aim of generating
a map in which the distances between markers are known with
reasonable accuracy. The various methods include restriction
mapping, in which fragments of DNA are generated by cutting with
restriction enzymes (also known as restriction endonucleases). The
recognition sequences for restriction enzymes are short (usually 4, 5 or
6 base pair) sequences that occur at defined positions in the DNA. By
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using combinations of restriction enzymes and working out the sizes of
the fragments, the ‘puzzle’ can be pieced together to give the pattern of
restriction enzyme recognition sites in the DNA. This is useful as defined
fragments can then be identified, either by size or by using a specific
DNA probe to bind to its complementary sequence. The technique of
restriction mapping is shown in Table 4.1.3 and Fig. 4.1.4.
Table 4.1.3: Fragments produced from a 15 kbp DNA fragment digested
with the restriction enzymes BamHI, EcoRI and PstI. Single, double and
triple digests are shown. All fragments are in kbp. These data can be
used to map the restriction sites as shown in Fig. 4.1.4.
BamHI
EcoRI
PstI
BamHI
EcoRI
BamHI
PstI
EcoRI
PstI
BamHI
EcoRI
PstI
14
1
12
3
8
7
11
3
1
8
6
1
7
5
3
1
6
5
3
1
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Fig. 4.1.4: Determination of the restriction map for the fragments listed
in Table 4.1.3. In (a) the BamHI site cuts the 15 kbp fragment into 14
and 1 kbp fragments. In (b) the EcoRI fragments of 12 and 3 kbp can be
orientated in two ways with respect to the BamHI site (i and ii). A
double digest with BamHI and EcoRI generates fragments of 11, 3 and 1
kbp, so the correct orientation is (b)ii, giving the pattern shown in (c).
Similar reasoning with the pattern of the PstI fragments shown in (d)
enables the final map to be determined (e). (Copyright D S T Nicholl/
Cambridge University Press. Reproduced with permission.)
Another method of identifying particular DNA sequences is to amplify
the target sequence using the polymerase chain reaction (PCR). This
technique has become very important in molecular biology, for both
mapping and identification of genes, and in forensic analysis. The basis
of the technique is shown in Fig. 4.1.5. In mapping, unique sequences in
the genome can be amplified by PCR if suitable primers are available.
This approach has become one of the most widely used for physical
mapping of genome fragments.
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Fig. 4.1.5: The polymerase chain reaction (PCR). The DNA duplex is
heat-denatured and two primers are annealed to the sequence. A DNA
polymerase then copies the templates, doubling the number of copies.
The denature-prime-copy cycle can be repeated automatically if a
thermostable polymerase (such as Taq polymerase) is used. The PCR
reaction generates a large number of copies of the target sequence in a
short time. (Copyright D S T Nicholl/Cambridge University Press.
Reproduced with permission.)
The various methods used in genome mapping enabled the
construction of a useful genetic and physical map of the human genome
by 1998. The pieces of DNA that are actually used for sequencing are
often cloned in large-capacity vectors that are essentially artificial
chromosomes. These can then be restriction mapped and sequenced.
The link between genetic, physical and restriction maps is shown in
Fig. 4.1.6.
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Fig. 4.1.6: Genome mapping. In (a) the genetic map is shown, with
genetic markers assigned to positions on the chromosome. In (b) a
section of the chromosome is shown, with the physical map of this
region. Various methods may be used to assign physical markers to their
chromosomal locations. In (c) the clone map of a section of the physical
map is shown, with large overlapping DNA fragments. From this a more
detailed restriction map and the DNA sequence itself can be determined.
DNA sequencing
The final stage of the genome project is to determine and assemble the
actual DNA sequence itself. There are several critical requirements for
this part – the DNA fragments must be generated, the sequencing
technology must be accurate and fast, and computer hardware and
software must be available to analyse the sequence data.
The technique used for sequencing is based on one of the original
methods developed in the mid 1970s. This is called the dideoxy chaintermination method, and relies on making a copy of the DNA template
to be sequenced. A DNA polymerase is used, along with a primer and
the four dNTPs (dATP, dGTP, dCTP and dTTP). With the correct
biochemical conditions, including a radioactive ‘label’ to enable the
product to be detected, the polymerase can make a copy of the DNA by
a process that is essentially the same as that used in DNA replication.
The chain termination part is what makes the key difference. This
involves setting up four separate reactions, each including one of the
four dideoxy NTPs (ddATP, ddGTP, ddCTP and ddTTP). These modified
nucleotides cannot form the next phosphodiester bond in the growing
chain – hence when a ddNTP is incorporated into the copy, it terminates
the process. The large number of fragments that are produced in the
four reactions produce a set of sequences that differ in length by one
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base, and end with a particular ddNTP. These fragments can be
separated by gel electrophoresis, and an image produced on X-ray film,
which is darkened by the radioactive label in the copied DNA strand.
The sequence is read from the film as shown in Fig. 4.1.7.
Fig. 4.1.7: Reading a sequencing gel. In (a), part of an X-ray film is
shown. The film was produced from the electrophoresis gel that
separated the DNA fragments in the direction of the arrow. In (b), a
tracing of the film is shown. A separate lane is used for each dideoxy
reaction, and the sequence can be read from the smallest fragment at
the bottom. (Autoradiograph courtesy of Dr N Urwin. Copyright D S T
Nicholl/Cambridge University Press. Reproduced with permission.)
The standard chain-termination method of sequencing was adapted for
genome sequencing by using fluorescent labelling instead of
radioactive methods. In one method, the ddNTPs can be tagged with
different labels, and one reaction carried out where all four ddNTPs are
used together. The products are separated by gel electrophoresis, and
the fluorescent labels detected as they come off the bottom of the gel.
This gives a direct readout of the sequence. The process can be
automated, and is much faster than conventional sequencing. It is
summarised in Fig. 4.1.8.
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Fig. 4.1.8: Automated DNA sequencing using fluorescent marker dyes.
Each ddNTP is tagged with a different dye as shown in (a). On
separation in a single lane of a sequencing gel, the DNA fragments pass
through a detector (b) and the fluorescent labels are monitored. A
computer captures the data and displays the sequence as a series of
peaks (c), from which the sequence is read as shown in (d).
Comparative genome analysis
As already mentioned, other genomes are being sequenced in addition
to the human genome. Whilst only a few have been completely
sequenced to date, the analysis of genomes has been carried out for a
number of years. Genetic and physical mapping, hybridisation studies
and sequencing of individual genes have provided a lot of useful
information about genome size and organisation. In fact, the emphasis
in molecular biology is changing as the amount of detailed information
about genes increases – we are now much more likely to consider the
function of a gene as part of the genome, rather than in isolation.
Some genome sizes are shown in Table 4.1.9. Size and complexity
generally increases with the increasing complexity of the organism, as
might be expected. Gene structure in bacteria is simpler than that in
eukaryotes. Comparing gene structure and organisation in E. coli, the
yeast S. cerevisiae and humans puts the complexity in the expected
order – bacterium, yeast, human. However, there are some anomalies –
many plants have much larger genomes than humans, for example. Thus
genome analysis is an area of research that is likely to give rise to many
unexpected discoveries in the next few years.
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Table 4.1.9: Genome size and estimated number of genes for some
organisms. Genome sizes are given in Megabase pairs
(1 Megabase = 1 × 106 bases).
Organism
Genome size
(Mb)
Number of
genes
Escherichia coli (bacterium)
4.6
4,405
Saccharomyces cerevisiae (yeast)
12.1
5,800
Drosophila melanogaster (fruit fly)
150
12,000
Homo sapiens (man)
3,000
70,000
Nicotiana tabacum (tobacco)
4,500
43,000
4.2 Human therapeutics
Congenital abnormalities are genetically-based diseases (often simply
called genetic diseases) that are present at birth. Diseases caused by a
single-gene defect are known as monogenic traits, and to date around
5,000 have been described. These are characterised as either autosomal
dominant, autosomal recessive or X-linked. Most X-linked traits are
recessive. Diseases that involve several genes are known as polygenic
traits, and are usually more difficult to diagnose and treat than the
single-gene defects. In this section we will consider how molecular
genetics has improved the diagnosis and treatment of genetic disease,
concentrating on two examples; cystic fibrosis and Duchenne
muscular dystrophy.
Detecting gene disorders
The characterisation of a monogenic genetic disease usually begins with
the presentation of the disease symptoms. Often a large amount of
historical information is available about the disease and its effects before
the definitive genetic cause is established. The first step is to trace the
disease through family relationships by carrying out pedigree analysis
to determine if the faulty gene is dominant, recessive or X-linked. This
information can often be useful in advising prospective parents about
the probability of their children inheriting the disease. This genetic
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counselling is an important part of preventive healthcare, and modern
molecular techniques have greatly improved the diagnosis of defective
alleles of the genes involved.
Once the disease has been identified as a monogenic trait, the search for
the gene defect itself can begin. This process can take many years to
complete, and often involves collaboration between many groups of
scientists specialising in the disease. The processes involved are similar
to those employed for the human genome project. The defect is firstly
mapped onto a chromosomal region by looking for genetic markers that
are co-inherited with the disease. Meiotic recombination frequency gives
an indication of how close the markers are to the target gene; the more
often the gene and marker are co-inherited, the closer they are on the
chromosome. When the chromosomal location of the gene is
established, the more detailed analysis of the region can begin. By using
a combination of genetic map information and physical mapping, the
defective gene can be tracked down, characterised and sequenced,
which can lead to the development of more accurate diagnostic
procedures and potential new treatments.
Cystic fibrosis (CF) is one disease that has been investigated in detail at
the genetic and molecular levels. CF is an autosomal recessive
monogenic trait that affects around 1 in 2,000 people. The CF gene
codes for a membrane carrier protein of 1,480 amino acids, which has a
complex structure with two transmembrane domains, two ATP-binding
domains, and a regulatory region. Mutations in the CF gene result in a
defective ion transport system which means that epithelial surfaces are
not fully hydrated. This causes sticky mucus accumulation in the lungs.
Major symptoms are inflammation of lung tissue and persistent bacterial
infection. Other symptoms can include defects in pancreatic function.
The disease is relatively easy to diagnose, as a symptom is the
production of salty sweat. Affected individuals have a reduced quality of
life and a life expectancy of about 30 years.
The defective gene for CF has a carrier frequency of around 1 in 22. In
cases where two carriers have children, there is a 1 in 4 chance of the
child receiving both defective alleles (one from each parent) and thus
being homozygous and suffering from the disease. The search for the CF
gene was a major undertaking, involving several research groups at
different stages. The gene was mapped onto chromosome 7, and linkage
studies with the gene and two types of physical marker narrowed down
the search. By an extensive programme of examining cloned DNA
fragments, using the techniques of chromosome walking and
chromosome jumping (see Fig. 4.2.1), the gene was eventually found in
1989.
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Fig 4.2.1: Chromosome walking and jumping. In (a) chromosome
walking is shown. The end of each overlapping fragment is used in a
hybridisation test to identify the next fragment. This is often used to
‘walk’ from a marker gene towards the target gene. Chromosome
jumping (b) is a similar technique but in this case a special cloning
technique is used to isolate complementary fragments that are far
apart. This enables a ‘jump’ along the chromosome, which is useful if
the marker gene is far from the target gene. Often a mixture of walks
and jumps is needed to progress, as shown in (b).
Once the gene had been identified, more and more details about the
molecular biology of CF began to emerge. Some 550 mutations have
been described, but the most common one by far is a deletion of three
base pairs that removes one amino acid from the polypeptide. This is
called the ∆ F508 mutation (∆ is deletion, F is phenylalanine and 508 is
the position in the protein). The defective protein does not fold up
properly, and does not reach its membrane location. A summary of the
CF gene, protein and the ∆F508 mutation is shown in Fig. 4.2.2.
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Fig 4.2.2: The cystic fibrosis ∆F508 mutation. In (a) the gene is shown.
Transcription produces the primary RNA transcript, that is converted
into the functional mRNA by removal of intervening sequences. On
translation the CF transmembrane conductance regulator protein
(CFTR) is produced. In (b) the normal and mutant proteins are shown.
Normal CFTR has phenylalanine (F) at position 508. In the mutant form
this is deleted, causing the protein to fold incorrectly.
Duchenne muscular dystrophy (DMD) is an X-linked disease that
affects around 1 in 3,300 boys. It causes progressive wasting of muscles,
resulting in wheelchair confinement by the teenage years and a life
expectancy similar to that for CF. There is a milder form of the disease
called Becker muscular dystrophy (BMD). As with the CF gene, a
search for the DMD gene was undertaken and it was found in 1987. The
protein (called dystrophin) is 3,685 amino acids in length! Its normal
function is to link the cytoskeleton with the sarcolemma (muscle cell
membrane) in muscle cells.
Molecular characterisation of genes enables accurate tests to be devised
for diagnosis of the conditions. The F508 deletion in CF can be
identified by amplifying a short (100 base pair) DNA fragment that spans
the area of the deletion. The polymerase chain reaction is used for this,
followed by separation of the DNA fragments on an electrophoresis gel.
In homozygous normal cases the two DNA fragments produced (one
from each allele) will be normal length and identical. The heterozygote
will show one shorter band in addition to the normal one, and the
homozygous recessive (two alleles with deletions) will show one band at
the lower position on the gel (Fig. 4.2.3). Molecular tests can be devised
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for most other gene mutations once the genes have been characterised,
and this area of molecular genetics has had a great impact on the
diagnosis of many types of inherited disease.
Fig 4.2.3: A PCR-based test for the cystic fibrosis (CF) defective allele. A
short sequence that spans the mutated region is amplified using the
polymerase chain reaction. In lane 1 a normal homozygous pattern is
shown (+/+). In lane 2 a carrier (heterozygous; +/∆F508) shows two
bands, one normal and one smaller by 3 nucleotides (one codon,
representing the deletion of phenylalanine). In lane 3 a CF-affected
individual (homozygous recessive; ∆F508/∆F508) shows one band at the
lower position. In lanes 1 and 3 the DNA band contains sequences from
both paternal and maternal chromosomes that run to the same position
in the gel. It is only in the heterozygous case that the two bands are
distinguished.
Gene therapy
When a gene defect has been identified and the gene cloned, there is
the possibility of using the ‘good’ copy of the gene to overcome the
problem. This is known as gene therapy. The proposition is attractive as
the cause of the disease is the target rather than just the symptoms.
Gene therapy might also be used to kill abnormal cells, or to inhibit the
spread of viruses by preventing DNA replication.
Although still in the early stages, some success has been achieved. In
1990 the first gene therapy procedure was used with a 4-year-old girl
patient who had the inherited disease adenosine deaminase (ADA)
deficiency. The defect in this enzyme leads to the condition known as
SCIDS (severe combined immunodeficiency syndrome), which means
that the patient cannot fight infection. By treating cells from the
immune system with a viral vector that carried the functional copy of the
gene, and replacing the cells in the patient, the condition was improved.
Cystic fibrosis and Duchenne MD are other obvious candidates for gene
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therapy. In both cases some success has been achieved using mice as
model organisms, although an effective therapy for use in humans is still
some way off due to the technical difficulties in getting the procedures
to work reliably. When assessing if gene therapy is likely to be
appropriate, several factors must be considered:
• the nature of the gene defect – the gene must be available in cloned
form, and the defective function characterised.
• the target cells in the patients – obviously the therapy must act on the
cells that are involved in the presentation of the disease. The cells can
be treated outside the body (ex vivo) and replaced, or may have to be
treated within the patient’s body (in vivo).
• the method of delivery of the normal gene – viral vectors can be used
but are not ideal. Other methods with potential are liposomes that
can fuse with cell membranes, or human artificial chromosomes.
• the expression and stability of the normal gene in the target cells –
the normal gene has to be expressed in the cells and the gene
product has to function properly.
One aspect of gene therapy that raises particular ethical questions
concerns the type of cells that are the targets. Most scientists and
observers accept that carrying out gene therapy on somatic cells (body
cells) is not much different from taking an aspirin – the chemical (DNA)
is just a little more complex. However, it is also possible that germ cell
gene therapy could be developed, in which the reproductive cells are
the targets. This would effectively alter the gene pool of the species, as
the alteration would be passed on to the next generation. At present,
there are no plans to carry out genetic manipulation of germ-line cells,
and most people agree that this should not be attempted.
4.3 Forensic uses
In the legal profession, the use of DNA technology has become one of
the most important tools for identifying individuals in both criminal
cases and in disputes over whether people are related or not (paternity
disputes and immigration cases are the most common). The original
technique was called DNA fingerprinting. Improvements in the
technology have increased the range of tests that can be carried out, and
today the more general term DNA profiling is used to describe the
methods available. The basis of all the techniques is that a sample of DNA
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from a suspect (or person in a paternity or immigration dispute) can be
matched with that of the reference sample (from the victim of a crime,
or a relative in a civil case). In scene-of-crime investigations, the
technique can be limited by the small amount of DNA available in
forensic samples. Modern techniques use the polymerase chain
reaction (see Fig. 4.1.5) to amplify and detect minute samples of DNA
from bloodstains, skin fragments or hair roots.
The original DNA fingerprinting technique is based on the fact that there
are highly variable regions of the genome that are specific to each
individual. These are minisatellite regions, which have a variable
number of short repeated-sequence elements known as variable
number tandem repeats (VNTRs). When digested with restriction
enzymes, different sized fragments are produced (Fig. 4.3.1). The
fragments can be detected using a probe that binds to the VNTR
sequence. This generates a unique profile of the DNA from that person.
Fig 4.3.1: Variation in restriction fragment lengths caused by different
numbers of tandem repeat elements. A locus that is heterozygous for the
tandem repeat length is shown. In the upper example, one allele with a
5-copy variable number tandem repeat (VNTR) is shown. In the lower
example, the allele has a VNTR with 8 copies. If these DNA molecules are
cut with a restriction enzyme that does not cut in the repeat sequence,
but cuts frequently outside this, the VNTR sequences will show as different length fragments (labelled 1 and 2 in the diagram; restriction sites
shown by arrows). A radiolabelled probe can then be used to identify
the repeat sequences as part of a DNA fingerprinting (profiling) experiment.
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Detecting fragments and producing a DNA profile using the original
techniques involves a number of stages:
• DNA isolation – this is carried out on the sample (often a blood
sample). If sufficient DNA is available it may be used directly in the
profiling technique.
• restriction enzyme digestion – this generates the DNA fragments that
produce the profile, with fragments of different lengths produced
from the variable regions of the DNA.
• gel electrophoresis – this separates the DNA fragments according to
length, and will produce the banding pattern that is used to compare
the different samples.
• blotting the DNA onto a filter – this enables the DNA fragment pattern
to be transferred to a filter for the hybridisation stage which detects
the target sequences.
• hybridisation with the probe – this involves using a labelled nucleic
acid probe (see Fig. 4.1c) which binds to a specific base sequence in
the target fragments.
The critical part is the selection of the appropriate probe sequence.
There are two main classes of these – multi-locus probes and singlelocus probes. Multi-locus probes bind to more than one site in the
sample, and give complex profile patterns. This can make the results
difficult to analyse, but it does decrease the possibility of a chance
match. This is obviously important in cases where legal decisions are
made on the strength of DNA fingerprint evidence. The odds against a
chance match for varying numbers of bands in a DNA profile are shown
in Table 4.3.2.
The use of multi-locus DNA profiling in a forensic case is shown in Fig.
4.3.3. In this example, blood from the victim is the reference sample.
Samples from seven suspects were obtained and treated along with the
sample from the victim. By matching the band patterns it is clear that
suspect 5 is the guilty party.
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Table 4.3.2: The odds against chance matches in a DNA fingerprint. The
more bands present, the less likely it is that any match is due to chance.
(Courtesy of Cellmark Diagnostics.)
Number of bands in fingerprint
Odds against a chance match
4
250 : 1
6
4,000 : 1
8
65,000 : 1
10
1 million : 1
12
17 million : 1
14
268 million : 1
16
4,300 million : 1
18
68,000 million : 1
20
1 million million : 1
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Fig. 4.3.3: A DNA profile prepared using a multi-locus probe. Samples of
DNA from the victim (V; boxed) and seven suspects (1–7) were cut with
a restriction enzyme and separated on an agarose gel. The fragments
were blotted onto a filter and a radioactive probe added. The probe
hybridises to the target sequences, producing a profile pattern on X-ray
film. The band patterns from the victim and suspect 5 match. (Copyright
Cellmark Diagnostics. Reproduced with permission.)
Single-locus probes will bind to just one complementary sequence in the
haploid genome. Thus, two bands will be visible in the resulting
autoradiogram; one from the paternal chromosome and one from the
maternal chromosome. This gives a simple profile that is often sufficient
to demonstrate an unambiguous match between the suspect and the
reference. The result of a paternity test using a single-locus probe is
shown in Fig. 4.3.4. Sometimes two or more probes can be used to
increase the number of bands in the profile. Single-locus probes are
more sensitive multi-locus probes, and can detect much smaller
amounts of DNA. Usually both single-locus and multi-locus probes are
used in any given case, and the results combined.
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Fig. 4.3.4: A DNA profile prepared using a single-locus probe for paternity testing. Samples of DNA from the mother (M), 4 children (1–4) and
the father (F) were prepared as in Fig. 4.3.3. A single-locus probe was
used in this analysis. The band patterns show two maternal bands and
two paternal bands. In the case of child 1, the paternal band is different
from either of the two bands in lane F, indicating a different father
(band labelled DF). This child was in fact born to the mother during a
previous marriage. (Copyright Cellmark Diagnostics. Reproduced with
permission.)
In forensic analysis, the original DNA profiling technique shown in Fig.
4.3.3 has now been largely replaced by a PCR-based technique that
amplifies parts of the DNA known as short tandem repeats (STRs, also
known as microsatellites). The PCR reaction overcomes any problems
associated with the tiny amounts of sample that are often found at the
crime scene. By using fluorescent labels and automated DNA detection
equipment (similar to the genome sequencing equipment shown in Fig.
4.1.7) a DNA profile can be generated quickly and accurately.
To ensure that results from DNA profile analysis are admissible as
evidence in legal cases, several important quality control steps must be
in place. These include accurate recording of the samples as they arrive
at the laboratory, and careful cross-checking of the procedures to make
sure that the test is carried out properly and that the samples do not get
mixed up. If PCR amplification is used as part of the procedure, great
care must be taken to ensure that no trace of DNA contamination is
present. A smear of the operator’s sweat can often be enough to ruin a
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test, so strict operating procedures must be observed, and laboratories
inspected and authorised to conduct the tests. This is essential if public
confidence in the technique is to be maintained.
4.4 Agriculture
Gene technology in agriculture has great potential, and can be applied
to both plants and animals. The aim is usually to achieve genetic
modification of the target organism to ‘improve’ some aspect that
would be of benefit to the producer or the consumer. Plants that are
resistant to disease, drought, herbicides and pesticides increase yields
and enable growth to be controlled more easily. Farm animals with
increased yields of milk and meat would seem to be a positive
development, as would increasing the shelf life of fruits and vegetables.
However, not all developments are accepted by the consumer, which
poses a problem for all concerned in the development, production and
marketing of genetically modified plants and animals.
Genetic modification usually involves inserting genes for the desired
characteristic into the host’s DNA to produce a transgenic organism,
which carries the new genetic material in a stable form that is expressed
and is also transmitted from generation to generation. An alternative use
of DNA technology is to produce substances such as growth hormones
by recombinant DNA methods, and use the product to affect the target
organism. We will consider both of these approaches by looking firstly at
transgenic plants, and then the production and use of bovine growth
hormone.
Transgenic plants
There are several key stages in the production of a transgenic organism,
but two requirements are of particular importance. These are (i) a
suitable vector system that will enable the cloned DNA fragment to be
inserted into the plant cell genome, and (ii) a mechanism for
regenerating whole plants in which all the cells carry the transgene. If
the latter is not achieved, a mosaic organism can arise, in which only
some of the cells have been modified. In plants, the techniques of tissue
culture can be used to regenerate whole plants from single cells (see
Fig.1.5.1) and thus mosaics are not usually a problem.
The most commonly used vector system for plant gene manipulation is
the Ti plasmid. This is found in the soil bacterium Agrobacterium
tumefaciens, and is responsible for causing crown gall disease in plants.
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This disease produces tumours at the base of the plant stem. The Ti
plasmid (Ti stands for Tumour inducing) is a large plasmid that carries a
region of DNA called T-DNA that can integrate into the plant cell
genome. This feature can be used to construct vectors that can be used
to deliver the target gene into the plant cell. The Ti plasmid itself is too
large to be used as a vector directly, as large plasmids are difficult to
handle in vitro without fragmenting. Thus smaller vector plasmids with
only the essential T-DNA region have been constructed. In many cases
these plasmids lack the tumour-forming characteristics of the intact Ti
plasmid, and thus do not cause tumours when regenerating the plant
from the genetically engineered cells. This makes it easier to produce
plants that are essentially normal apart from the inclusion of the
transgene. A summary of the method for producing a transgenic plant is
shown in Fig. 4.4.1.
Fig. 4.4.1: Generating a transgenic plant. In (a), the Ti-based vector is
prepared from the host strain of Agrobacterium tumefaciens (although
often E. coli is used as the host at this stage). The vector is cut with a
restriction enzyme (RE). In (b), the gene of interest is isolated and
inserted into the vector to generate a recombinant plasmid. This can be
transferred into plant cells in culture, either by direct introduction or
by using Agrobacterium infection to deliver the gene. Plants with the
gene are regenerated from tissue culture, and can then be used in
standard breeding procedures.
An example of the use of transgenic plant technology is the construction
of transgenic tomato plants that have been engineered to delay the
ripening process. In the transgenic plant the production of ethylene
(which causes ripening) is inhibited, and thus the fruit stays firmer for
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longer. This technology was used by the biotechnology company
Calgene to produce the Flavr Savr (sic) tomato. Another example is the
incorporation of insect resistance into plants by inserting a gene that
produces an insecticidal protein. In one case a gene from the soil
bacterium Bacillus thuringiensis was inserted into tomato and tobacco
plants. When insect pests feed on the plants expressing the gene, the
protein is converted into an insecticidal toxin by enzymes in the insect’s
gut. Other animals do not have this enzyme, and thus are not affected by
the toxin.
A major goal of plant genetic modification is the introduction of
nitrogen fixation genes into non-leguminous crops, which lack the root
nodules that contain the nitrogen-fixing bacteria Rhizobium spp. One of
the problems is that the Ti-based vector system does not infect monocot
plants such as cereals and grasses, many of which are the main target
crops. However, other methods of introducing genes into these crops
are being used with some success.
One of the main controversies in plant genetic modification arose over
the production of ‘Roundup ready’ soybean plants by the biotechnology
company Monsanto. These plants were modified to be resistant to the
herbicide glyphosate, which is the active component of the commercial
weedkiller Roundup. The benefits of this are not in question – farmers
can use this broad-spectrum herbicide to control weeds without
affecting the modified crop plants. However, the main concern has
centred on the containment of the modified plants. There are strict
guidelines to regulate how such plants can be grown, and several cases
have been reported where these guidelines were breached. Many
people are concerned that herbicide resistance may spread to other
species, with potentially serious consequences in the medium to long
term. This is an area of active debate at present, and much more careful
work is needed to reassure the public that genetically modified crops
are safe for both consumption and in terms of their environmental
impact.
Bovine growth hormone
Agricultural animals are also targets for genetic modification, and many
types of transgenic animals have been constructed. As with plants, there
are concerns about the use of this type of modification, both in terms of
the technical aspects and also in the area of animal welfare, and this is
again an area of heated debate. However, one application of gene
technology in agriculture is already well established, and is different in
that it involves a genetically engineered product that is administered to
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farm animals. Examples include vaccines and antibodies, as well as
growth hormones that can be used to increase growth and milk
production in cattle. Tests have also been carried out with recombinant
cellulase, which hydrolyses cellulose and therefore improves the
digestibility of animal feed that includes plant material.
The growth hormone bovine somatotrophin (BST) has been available
for several years from Monsanto. The gene for BST has been cloned into
a bacterial system where it is expressed during bacterial growth. The
product is then purified and prepared for administration to cattle, either
by injection or by including the protein in animal feed. The production
of recombinant BST is outlined in Fig. 4.4.2. The effects of BST are to
increase milk production by around 10%, which is obviously attractive to
farmers. In strictly technical terms the result is not harmful to humans,
as any ingested protein will be digested in the gut and will not have any
effect on the consumer. However, as in the case of crop plants, there is
widespread public concern over the use of this technology, with some
countries refusing to import meat or milk from BST-treated cattle.
Fig. 4.4.2: The production of bovine somatotrophin (BST). In (a), the
vector plasmid is prepared and cut with a restriction enzyme. In (b), the
gene for BST is isolated from cattle and ligated into the vector to produce a recombinant. This is replaced into E. coli cells, where BST protein
is synthesised. This can then be produced commercially using largescale fermentation processes as shown in (c).
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The future?
Despite the many useful advances that have been made in applying gene
manipulation techniques to agriculture, at the end of the 1990s a very
strong feeling of unease developed in some parts of the scientific
community and within the general public. This centred on the problems
associated with transgenic crop plants for human consumption. Terms
such as ‘Frankenstein foods’ have been used, which tend to
sensationalise the problems rather than address them objectively.
Animal welfare issues associated with transgenic farm animals have also
provoked heated debate, and many people have called for a halt to
developments. Such caution is not new in DNA technology – in the
1970s scientists were concerned about the spread of potentially harmful
characteristics among bacterial populations. What is different is that now
the public are more aware of the impact that gene manipulation is
having on our lives and on the environment.
Perhaps the greatest challenge for scientists in the new millennium is to
use DNA technology in a way that is acceptable to everyone concerned.
Towards the end of 1999 many supermarkets were claiming that they
would not stock genetically modified foods, and this is a very real
problem for the companies involved in the production and use of
transgenic crop plants. In many ways the problems that lie ahead in
genetic modification are the social and ethical aspects of the technology,
rather than technical difficulties. What is certain is that the debates will
continue, and that we need to increase public awareness of both the
benefits and the possible problems associated with gene manipulation
and its applications.
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FURTHER READING
Campbell, N A et al, Biology (5th edn), Menlo Park and Harlow:
Addison-Wesley, 1999
Raven, P H and Johnson, G B, Biology (5th edn), Boston and London:
WCB/McGraw-Hill, 1999
Two popular general biology texts that have proved their worth
through numerous editions. Both provide excellent coverage of the
topics in the Unit, and also cover many other aspects in the Advanced
Higher Course. Excellent value for money. Additional educational
aids are available.
Alberts, B et al, Essential Cell Biology: An Introduction to the
Molecular Biology of the Cell, New York and London: Garland
Publishing, 1998
An excellent shorter version of the classic Molecular Biology of the
Cell. Covers all elements of the Advanced Higher Biology Unit in Cell
and Molecular Biology. Very clearly written and illustrated.
Brown, T A, Genomes, Oxford: Bios Scientific Publishers, 1999
A new text which approaches the subject of molecular genetics from
the perspective of the genome rather than a more traditional
treatment. Detailed coverage of genome structure and function,
including mapping and sequencing.
Sudbery, P, Human Molecular Genetics, Harlow: Longman, 1998
A detailed text dealing with aspects of human genetics, including
disease, mapping and genome sequencing.
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GLOSSARY
α -helix A right-handed helical form of secondary structure in proteins.
actin A protein found in muscle fibres and other contractile
components such as microfilaments.
active transport The movement of substances across a membrane
against the concentration gradient. This process requires energy.
ADA Adenosine deaminase, an enzyme involved in nucleotide
metabolism. ADA deficiency causes SCIDS q.v. ADA deficiency was one
of the first targets for gene therapy.
adenine Nitrogenous base found in DNA and RNA. Forms A:T base pairs
with thymine.
adenylate cyclase An enzyme which catalyses the formation of cyclic
adenosine monophosphate (cAMP) from ATP. Involved in signal
transduction. Sometimes known as adenyl cyclase.
adrenaline A hormone of the catecholamine group, which has a variety
of effects such as speeding up heartbeat and stimulating the
breakdown of glycogen to glucose – these effects are sometimes
called the ‘fight or flight’ response. Adrenaline can also act as a
neurotransmitter. Also known as epinephrine.
Agrobacterium tumefaciens A bacterium that is used in gene
manipulation of plants. It carries the plasmid responsible for crown
gall disease.
allosteric modulator (effector) An effector that binds to a site on an
enzyme, usually distinct from the active site, to regulate its activity.
amino acid The monomeric unit of proteins. Made up of an amino
group, carboxyl group and a hydrogen atom attached to a carbon
known as the α-carbon. The fourth group is an R-group, which gives
the amino acid its characteristics. There are 20 amino acids found in
proteins.
amino terminus (N-terminus) The end of a polypeptide chain which
has the free amino group.
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anabolic reaction A ‘building up’ or biosynthetic reaction. Opposite of
catabolic.
anticodon The triplet of bases on a tRNA molecule that is
complementary to the codon in mRNA.
antiparallel Describes the orientation of the strands in a DNA duplex;
these run in opposite directions with respect to their 5'-3' polarity.
antiproliferation genes Also known as tumour-suppressor genes, these
are involved in controlling (restricting) cell division activity. When
antiproliferation genes are defective, tumours may form.
ATP Adenosine triphosphate. Made up of the sugar ribose and the base
adenine (the nucleoside adenosine) and three phosphate groups (the
triphosphate). ATP is one of the most important molecules in cell
metabolism, as it is used as an energy transfer compound.
ATPase An enzyme that hydrolyses ATP to produce ADP and energy.
autosome A chromosome that is not a sex-determining chromosome.
We have 22 pairs of autosomes and one pair of sex chromosomes.
β -sheet Type of secondary structure in proteins where the polypeptide
chains are arranged as parallel ‘sheets’, unlike the α-helix.
batch culture Method of growing cells in culture where the volume of
the culture is fixed.
bioinformatics Applies to the gathering, storage and analysis of
biological information. Usually refers to DNA sequence information
generated by genome sequencing.
blastula The hollow ball of cells formed after several rounds of cell
division following fertilisation.
bovine somatotrophin (BST) Growth hormone from cattle. Produced
by recombinant DNA technology and available for administration to
cattle to improve yield.
C-terminus See carboxyl terminus.
callus An undifferentiated mass of cells growing in tissue culture.
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cancer cell A cell that has become transformed to grow in an
unrestrained way, often due to defective cell cycle control processes.
carbohydrates Important metabolic compounds made up of carbon,
hydrogen and oxygen. Monomers are the monosaccharides q.v., from
which large polysaccharides can be synthesised.
carboxyl terminus (C-terminus) The end of a polypeptide chain which
has the free carboxyl group.
carrier protein A membrane-bound protein that can bind other ions or
molecules and transport them across the membrane by either
facilitated diffusion or active transport.
catabolic reaction A ‘breaking down’ reaction (opposite of anabolic).
The breakdown of glucose in respiration is an example of an
important catabolic pathway.
catalytic cycle The bind-catalyse-release cycle of events that enable
enzymes to perform their catalytic functions.
cell cycle The period between the formation of a cell and its division to
form two new cells. Has four stages: G1, S, G2 and M.
cell signalling General term to cover the generation, reception,
transduction and effect of various types of molecules that act as interor intracellular signals.
cellobiose Disaccharide composed of two glucose molecules joined
together by a β(1,4) linkage.
cellulase An enzyme that hydrolyses cellulose.
cellulose Polysaccharide made up of glucose monomers joined by
β(1,4) linkages. Found in cell walls of plants, it is the most abundant
organic compound in the biosphere.
centriole Microtubule-based structure found mostly in animal cells as a
pair of centrioles, important in organising spindle fibres during cell
division. The centriole is also part of the structure of cilia and flagella.
centromere The constricted region of a replicated chromosome where
the two chromatids are held together.
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centrosome Structure found in eukarytoic cells that is involved in the
organisation of microtubules, particularly during cell division. Cf.
microtubule organiser.
channel protein Type of transport protein that forms a channel across a
membrane, through which substances can pass.
chitin Polysaccharide found in fungal cell walls and insect exoskeletons,
composed of N-acetylglucosamine units.
chloroplast Organelle found in plant and algal cells. Highly organised
internal structure of membranes, on which the light reactions of
photosynthesis are localised. The dark reactions occur in the nonmembrane region or stroma.
cholesterol Steroid found in cell membranes. Also the basis of many
other steroid hormones.
chromatid Refers to the copy of a chromosome after DNA replication,
prior to cell division. Each replicated chromosome is composed of
two chromatids, joined at the centromere.
chromosome A DNA molecule carrying genetic information. In
prokaryotic cells there is a single circular chromosome. In eukaryotic
cells there are multiple linear chromosomes (46 in man), associated
with histone and non-histone proteins.
codon Triplet of bases in DNA or mRNA which specifies an amino acid or
a stop signal.
complementary Refers to pairs of strands in a double-stranded nucleic
acid molecule that bind together by base pairing.
condensation Refers to a reaction in which two molecules are joined
together by the removal of specific groups. Dehydration is a form of
condensation synthesis where water is removed.
conjugated protein A protein which has a non-protein component
associated with it. Examples include glycoproteins, lipoproteins and
nucleoproteins.
cortisol A steroid hormone that can diffuse across the plasma
membrane and act on gene regulatory proteins to stimulate
transcription.
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covalent modification A method of regulating enzyme activity by
adding/removing groups such as a phosphate group.
cyclic AMP (cAMP) Cyclical form of AMP, often formed from ATP by the
action of adenylate cyclase. Acts as a signalling molecule or second
messenger.
cystic fibrosis (CF) Disease in which ion transport across membranes is
affected in individuals who are homozygous for the defective allele.
Some 550 mutations in the CF gene have been described. CF is the
most common monogenic disorder in Western societies, with around
1 in 2,000 affected. Carrier frequency is approximately 1 in 22.
cytokinesis The separation of the cytoplasm during cell division.
cytoplasm The contents of a cell apart from the nucleus. Includes the
fluid cytosol and the cell organelles.
cytosine A nitrogenous base found in DNA and RNA. Forms G:C base
pairs with guanine.
cytoskeleton The network of structural fibres in the cell, often
associated with the cell membrane. The cytoskeleton is important for
the maintenance of cell shape.
cytosol The fluid-based part of the cytoplasm, excluding the organelles.
dehydration Removal of the elements of water from a reaction in which
two molecules are joined together. A specific type of condensation
reaction.
dictyosome Name sometimes used to describe the Golgi apparatus q.v.
in plant cells.
disulphide bond Covalent bond between two sulphur atoms. In
proteins, disulphide bonds are formed between the sulphydryl (-SH)
groups of cysteine residues in the polypeptide chain. Important in
stabilising the 3-D structure of proteins.
DNA Deoxyribonucleic acid (infrequently deoxyribosenucleic acid is
used). DNA is a polynucleotide consisting of two complementary
strands, base paired A:T and G:C. The genetic material in most
organisms.
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DNA fingerprinting (profiling) Technique which enables identification
of individuals based on variations in the pattern of restriction
fragments or by polymerase chain reaction q.v. amplification of
specific sequences. The original technique gives a ‘bar code’ result
that is unique to each individual.
DNA ligase Enzyme which catalyses the formation of phosphodiester
bonds between adjacent nucleotides in a polynucleotide. Used in
DNA replication and repair processes in vivo, it is also an essential
part of recombinant DNA technology, where it is used as ‘molecular
glue’.
DNA replication The copying of the genetic material prior to cell
division. Each strand of the double helix is used as a template for the
formation of a new strand; replication is therefore semi-conservative
in that the products contain one original and one new strand of DNA.
DNA replication occurs during the S (synthesis) phase of the cell
cycle.
DNA sequencing Determination of the order of bases in a DNA strand.
Various methods are available, the most commonly used being the
dideoxy chain termination method. Automated DNA sequencing has
enabled great progress to be made in sequencing whole genomes.
domain In proteins, a region of defined 3-D structure. A single
polypeptide chain may contain one or more domains, each folding
into a separate region of tertiary structure.
double helix The term used for the double-stranded DNA molecule,
usually to describe the right-handed B-form of the helix.
Drosophila melanogaster The fruit fly, an organism that has been used
extensively in genetic research for classical, molecular and
developmental genetic studies.
EGF Epidermal Growth Factor, a local mediator signalling molecule.
Stimulates cell division in epidermal and other cell types.
electron microscope Complex (and expensive!) microscope which
uses electron beams as the ‘illumination’ source. Due to the short
wavelength, much higher resolution (and hence magnification) can
be achieved compared with light (optical) microscopy.
Electromagnetic lenses are used to focus the beam of electrons, and a
vacuum is required in the microscope body. Specimens must be dead
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and cut very thinly for transmission electron microscopy. Scanning
electron microscopes generate 3-D images of the specimen.
endocrine system The system of glands (endocrine glands) which
facilitates cell–cell signalling by secretion of hormones into the
bloodstream. Cf. paracrine system.
endomembrane system The system of internal membranes in
eukaryotic cells. Usually refers to the endoplasmic reticulum, Golgi
apparatus and plasma membrane with associated membrane-bound
vesicles such as lysosomes. Chloroplasts and mitochondria are not
considered part of the endomembrane network in cells.
endoplasmic reticulum (ER) Part of the endomembrane system in
eukaryotic cells. Consists of flattened sacs of membrane, linked to
give a complex network of channels. May be smooth (SER; lacks
ribosomes) or rough (RER; has ribosomes associated with the outer
surface of the membranes).
end-product inhibition Inhibition of an enzyme early in a metabolic
pathway q.v. by the final product of the pathway. A form of negative
feedback q.v.
enzyme A biological catalyst, specific for a particular reaction. Usually
protein molecules, often with associated non-protein components
such as metal ions. Some RNA molecules can act as enzymes, and are
known as ribozymes.
enzyme-linked receptor Type of receptor found in membranes, which
has an enzyme activity (often a kinase activity) associated with the
cytoplasmic part of the receptor. This generates a response in the
cytosol when the signal molecule binds.
epidermal growth factor (EGF) See EGF.
Escherichia coli Bacterium that has been a major research organism in
microbial genetics and biochemistry. Thousands of different strains of
E. coli are available.
eukaryote (eukaryotic) Applies to cells (or organisms) in which there
is a membrane-bound nucleus.
explant Piece of tissue (usually applies to plants) prepared for growth
under tissue culture conditions.
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extrinsic protein A protein associated with the outer surface of the
plasma membrane.
facilitated diffusion Transport of molecules or ions across a membrane
by a carrier protein with the concentration gradient. Does not require
energy.
fatty acid A long-chain organic acid (functional group COOH) that is a
component of triacylglycerols and phospholipids. The general
formula is CH3(CH2)nCOOH for saturated fatty acids.
fermentation Anaerobic carbohydrate catabolism. The term is often
used to describe the production of carbon dioxide and ethanol by
microorganisms such as yeast.
fibrous protein A protein which forms fibres and has a structural rather
than enzymatic function. Examples include keratin and collagen. Cf.
globular protein.
fimbriae Projections from the surface of a bacterial cell. Cf. pili.
flagella Thread-like structures, longer than fimbriae, involved in
motility. Found in bacteria and also unicellular eukaryotes.
fluid mosaic model The accepted model for membrane structure, in
which proteins are embedded or associated with a phospholipid
bilayer.
foetal bovine serum (FBS) Calf serum, used as a supplement in growth
media for animal cells in tissue culture.
functional group The defining group of a molecule, such as COOH,
NH2, C=O, etc.
GABA γ-aminobutyric acid, an inhibitory neurotransmitter.
gastrulation Stage in embryological development formed by
invagination of the blastula.
gene Unit of genetic information which generates an RNA molecule. This
may be mRNA (which is translated to give a protein) or may be tRNA
or rRNA.
gene manipulation Term used to describe the methods of recombinant
DNA technology.
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gene regulatory protein A protein which binds to DNA to regulate or
control gene expression.
gene therapy Use of a gene sequence as a therapeutic agent to attempt
to overcome the effects of a defective gene, as in ADA deficiency and
cystic fibrosis.
genetic mapping Method of determining the relative positions of genes
on a chromosome by analysing recombination frequencies. Cf.
physical mapping.
genetic modification Alternative term for gene manipulation, often
used where the aim is to alter the genetic makeup of an organism
rather than simply to clone a gene or other DNA sequence.
genome The genetic material of a cell or organism. In eukaryotes, can
be used to describe nuclear, mitochondrial and chloroplast DNA.
germ cell A reproductive cell as opposed to a somatic or body cell.
globular protein Protein which folds to give a complex 3-D tertiary
structure. Usually part of an enzyme. Cf. fibrous protein.
glucose Important hexose monosaccharide of the formula C6H12O6.
Glucose catabolism is a major source of energy generation in the cell.
glycerol Sugar alcohol with 3 carbons, forming the backbone of
triacylglycerols and phospholipids.
glycogen A storage polysaccharide found in animal cells. Composed of
branched chains of glucose molecules.
glycolysis A major pathway of primary carbohydrate catabolism where
glucose is broken down to pyruvate. The term literally means ‘sugar
splitting’.
glycoprotein A protein with sugars attached to specific amino acid
residues.
glycosaminoglycans Polysaccharides made up of repeating amino sugar
disaccharides.
glycosidic bond The covalent bond joining two sugars together.
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Golgi apparatus or complex Structure in eukaryotic cells involved in
modifying and packaging materials such as proteins. Consists of
flattened sacs of membrane.
G-protein-linked receptor Class of receptor based on the seven-pass
transmembrane protein, which has a G-protein binding site on the
intracellular side of the membrane.
Gram stain Method of staining bacterial cells which distinguishes cells
on the basis of their cell wall structure.
granum (pl. grana) Structural component of chloroplasts, composed of
a stack of flattened membrane sacs known as thylakoids.
growth factor Applies to any substance, other than a carbon source,
which is needed for growth.
guanine A nitrogenous base found in DNA and RNA. Forms G:C base
pairs with cytosine.
haeme group Iron-containing prosthetic group found in proteins such
as myoglobin and haemoglobin. Involved in the transport of oxygen.
haemoglobin Blood protein involved in carrying oxygen. Composed of
4 subunits, 2 α and 2 β chains, each with its own haem prosthetic
group.
heteropolymer A polymer in which the monomeric units are different;
the best examples are protein molecules, made up of 20 different
amino acid monomers.
heterotroph An organism that requires an organic carbon source. Cf.
photoautotroph.
histamine Amine derived from histidine. Acts as a local mediator and is
involved in inflammation and allergy responses.
homopolymer A polymer in which the monomeric units are all the
same; e.g. polyphenylalanine.
hormone Chemical signalling molecule that is made in one tissue or
gland, and transported to its site of action (another tissue) in the
bloodstream.
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human genome project The international project to determine the
base sequence of the human genome. Scheduled to be almost
completed by mid-2000, the entire sequence of the 3 billion base
pairs should be known by 2003.
hybrid Can be used to describe cells that are fused to generate a new
cell line in tissue culture.
hybridisation In molecular biology, refers to the annealing of
complementary nucleic acid sequences. Used to identify genes and
other DNA fragments.
hydrolase An enzyme that catalyses a hydrolytic reaction.
hydrolysis The splitting apart of two molecules by the addition of the
elements of water; the reverse of dehydration synthesis.
hydrophilic Literally ‘water-loving’; refers to molecules or parts of
molecules that are attracted to an aqueous environment, such as the
phosphate/nitrogen group of phospholipids.
hydrophobic Literally ‘water-hating’; hydrophobic molecules, such as
the fatty acid tails of phospholipids, avoid or exclude water.
hydrophobic interactions Attractions between hydrophobic R-groups,
important in determination of the tertiary structure of proteins.
immortalised cell line A cell line that is able to continue cell division
indefinitely when grown in tissue culture.
induced fit Alteration of the shape of an enzyme on binding a substrate,
which enhances the binding of the substrate and action of the
enzyme. Cf. lock and key.
inositol triphosphate Intracellular signalling molecule or second
messenger, produced by the action of phospholipase C.
insulin Hormone involved in the regulation of blood glucose levels.
intermediate filaments Elements of the cyoskeleton (others are
microfilaments and microtubules), composed of fibrous protein
strands.
intrinsic protein A protein that is fully embedded in a membrane;
sometimes called a transmembrane protein.
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intron (intervening sequence) Region of a gene that does not code for
the protein. Found in eukaryotic genes, introns account for the very
large size of some genes. The are removed by RNA processing after
transcription of the gene region.
ion-channel-linked receptor Class of receptor that forms a pore across
a membrane. May be permanently open or gated, where opening the
channel requires a specific stimulus.
isomers Compounds with the same number and type of atoms but
different structures.
isomerase An enzyme that catalyses an isomerisation, causing a change
to the structure of a molecule.
kinase An enzyme that adds a phosphate group, derived from ATP, onto
another molecule.
Krebs cycle Biochemical pathway for the complete oxidation of
pyruvate. Also known as the citric acid cycle or tricarboxylic acid
cycle.
lac operon The cluster of genes encoding the enzymes needed for
lactose catabolism in bacterial cells.
lactose A disaccharide composed of glucose and galactose.
light microscope Microscope with glass lenses that uses visible light as
the illumination source. Also known as an optical microscope.
lipid Molecule, usually classed with the macromolecules, that is soluble
in an organic solvent but insoluble in water. A diverse range of
molecules including the triacylglycerols, phospholipids and steroids.
lipoprotein A protein with a lipid molecule attached.
lock and key Model of enzyme action in which the substrate fits exactly
into a ‘pocket’ in the enzyme. Although essentially correct, the
induced fit model q.v. is a better representation of what actually
happens.
lysosome Sub-cellular membrane-bound organelle containing hydrolytic
enzymes involved in intracellular digestion.
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macromolecule A molecule made up of monomeric units joined
together by dehydration synthesis. Polysaccharides, proteins and
nucleic acids are the three main types found in cells. Lipids, although
smaller, are often classed as one of the four groups of intracellular
macromolecules.
maltose Disaccharide composed of two glucose molecules. Usually
produced by the hydrolysis of starch, and further hydrolysed by
maltase to give glucose.
messenger RNA (mRNA) The RNA molecule that carries information, in
the form of codons, produced by transcription of DNA. Cf. ribosomal
RNA, transfer RNA.
metabolic pathway A sequence of biochemical reactions that produces
a particular product or products. May be controlled by end-product
inhibition q.v.
microbodies Diverse class of small membrane-bound sub-cellular
organelles, including peroxisomes and glyoxysomes.
microfilaments One of the three types of filament found in the
cytoskeleton. Composed of the protein actin. Cf. intermediate
filaments, microtubules.
microtubule organiser Structure in a eukaryotic cell that controls
microtubule formation. Cf. centrosome.
microtubules Filamentous hollow strands composed of the protein
tubulin. With microfilaments and intermediate filaments they make up
the cytoskeleton. Also involved in spindle formation for cell division.
microvilli Projections from the surface of epithelial cells, particularly
gut cells.
middle lamella The layer between two adjacent plant cell walls.
mitochondrion (pl. mitochondria) Sub-cellular membrane-bound
organelle involved in respiration. The inner mitochondrial membrane
is folded to increase the surface area available for the localisation of
the reactions of the electron transport chain.
mitosis Cell division process in which chromatids (components of a
replicated chromosome) are separated prior to cytokinesis q.v.
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mitosis promoting factor (MPF) Protein complex involved in
controlling the entry of cells into mitosis during the cell cycle.
Activation of MPF is triggered by a rise in the concentration of cyclin,
another cell cycle protein.
monogenic Applies to a genetic trait that is caused by a single gene, as
opposed to a polygenic trait, in which there may be many genes
involved.
monolayer Refers to a layer of cells, one cell deep, growing in a flask or
dish.
monomer The ‘building block’ of polymers; examples are amino acids
(polymers are proteins) and nucleotides (polymers are nucleic acids).
monosaccharide Literally ‘single sugar’. Monosaccharides have the
formula (CH 2O)n. The simplest group are the trioses, with three
carbon atoms.
mosaic An organism whose cells have two or more genotypes. In
transgenic (q.v.) organisms, refers to the condition where not all the
cells carry the transgene.
motif A particular structural feature of proteins or nucleic acids.
Important in regulation of gene or enzyme activity.
mucilaginous capsule Outer layer of some bacterial cells, composed of
polysaccharide-based sticky material.
multi-locus probe In genetic fingerprinting, a probe that binds to more
than one sequence in the sample.
muscular dystrophy (MD) An X-linked disease affecting young boys
(frequency around 1 in 3,300), causing muscle wasting. Two variants
are Duchenne MD and Becker MD.
myoglobin Oxygen-carrying haeme protein composed of a single
polypeptide chain.
N-terminus See amino terminus.
NAD(H) Co-enzyme involved in oxidation/reduction. Important in
energy transfer reactions in the cell, the reduced form can be
‘exchanged’ for ATP (q.v.) in aerobic respiration via the electron
transport chain.
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negative feedback General term for control of a process where
detection of high levels of a product causes a reduction in its
production. Occurs in homeostatic physiological mechanisms and
also in biochemical pathways as end-product inhibition q.v.
neoplastic Refers to cells or tissues that arise as a result of uncontrolled
cell growth, often causing cancers in animals.
neuronal Type of communication involving nerve cells and
neurotransmitters.
neurone A nerve cell.
neurotransmitter A chemical substance that transmits an electrical
signal across the synapse in nerve cells. Examples include
acetylcholine and noradrenaline.
nitrogenous base A nitrogen-containing base found in nucleic acids.
Chemically can be either a purine (double-ring structure; e.g. adenine
and guanine) or a pyrimidine (single-ring; e.g. cytosine, thymine and
uracil).
nuclear envelope The double membrane surrounding the eukaryotic
nucleus.
nuclear magnetic resonance (NMR) A technique for determining the
3-D structure of small proteins and other molecules.
nuclear membrane See nuclear envelope.
nuclease An enzyme that hydrolyses phosphodiester bonds in nucleic
acids. May be an endonuclease (cuts within a nucleic acid strand) or
an exonuclease (cuts from one end). Ribonucleases digest RNA,
deoxyribonucleases digest DNA. The restriction endonucelases are
one important group.
nucleic acid General term used to describe DNA and RNA.
nucleoid Region in a prokaryotic cell where the DNA is localised; not a
true membrane-bound nucleus.
nucleoprotein A complex of protein and nucleic acid.
nucleoside A sugar joined to a nitrogenous base.
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nucleosomes Structures composed of histone proteins that are
involved in packaging DNA in chromosomes. Nucleosomes look like
beads on a string when seen in the electron microscope.
nucleotide A sugar, base and phosphate.
nucleus Membrane-bound compartment in eukaryotic cells. The DNA is
located in the nucleus, which is also the site of DNA replication and
transcription.
oncogene A gene involved in cancer. The normal version of an
oncogene is called a proto-oncogene.
optical microscope See light microscope.
organelle Sub-cellular membrane-bound structure in eukaryotic cells.
oxidoreductases A class of enzymes involved in oxidation/reduction
reactions.
paracrine system Intercellular signalling system involving short-range
effects. Cf. endocrine system.
passive transport Transport of substances across a membrane without
the need for energy expenditure.
pedigree analysis Method of retrospective genetic analysis. Involves
tracing inherited traits through family trees. Can enable predictions
to be made about the likelihood of parents passing on genetic defects
to their children.
peptide bond The C–N bond linking two amino acids together. Formed
by dehydration of the amino and carboxyl groups of adjacent amino
acids.
peptidoglycan Component of the bacterial cell wall, composed of
polysaccharide chains linked by short peptides.
peripheral protein A protein associated with the outer layer of the
plasma membrane. Sometimes called an extrinsic protein q.v.
peroxisomes Sub-cellular membrane-bound organelles which contain
catalase and peroxidases. Sometimes called microbodies q.v.
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phosphatase Enzyme that removes phosphate groups from molecules.
phosphatidylcholine A phospholipid which is the major component of
cell membranes.
phosphodiester bond The bond linking two nucleotides together via a
5'-3' linkage, formed between the 5' phosphate of one nucleotide and
the 3' hydroxyl of the next.
phospholipase C Enzyme that catalyses the formation of the second
messengers (q.v.) inositol triphosphate (IP3) and diacylglycerol (DAG)
from membrane lipids.
phospholipid Type of lipid found in cell membranes. Based on
triacylglycerol structure, with one of the fatty acid chains being
substituted by a phosphate. This gives the molecule a hydrophobic/
hydrophilic polarity.
phospholipid bilayer Two layers of phospholipid arranged with the
hydrophobic fatty acid ‘tails’ together. This gives a layer with a
hydrophobic interior and a hydrophilic surface, which is the basis of
cell membrane structure.
photoautotroph An organism that can synthesise its requirements from
inorganic carbon (CO2) and light. Cf. heterotroph.
photosynthesis The use of light energy to fix and reduce carbon
dioxide into sugars. Carried out in the chloroplasts of algae and
plants, and also by certain bacteria.
physical mapping Applies to a variety of techniques to determine the
positions of genes on a chromosome by locating a physical marker.
Cf. genetic mapping.
pili Projections from the bacterial cell surface. Cf. fimbriae.
plasma membrane The membrane surrounding the cell. Sometimes
known as the cell membrane or plasmalemma.
plasmodesmata Cytoplasmic connections across cell walls in plants,
linking adjacent plant cells.
pluripotent Having the potential to develop into a number of different
cell types, but not all the types that are found in the adult organism.
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polygenic Refers to a genetic trait that involves a number of genes. Cf.
monogenic.
polymer A large molecule (macromolecule) composed of monomeric
units joined together, usually by dehydration synthesis.
polymerase An enzyme that synthesises a polymer. Examples include
DNA and RNA polymerases, which are template-dependent
polymerases.
polymerase chain reaction (PCR) Method of amplifying DNA
sequences by repeatedly copying the region between two primers.
polynucleotide Polymer composed of nucleotides linked by
phosphodiester bonds.
polypeptide Polymer composed of amino acids linked by peptide
bonds.
polysaccharide Polymer composed of monosaccharides linked by
glycosidic bonds.
primary cell culture Cell culture derived from cells taken directly from
a tissue sample.
primary structure In proteins, the sequence of amino acids in a
polypeptide chain.
prokaryote Cell without a membrane-bound nucleus.
proliferation genes Genes that encode proteins which promote cell
division. Cf. antiproliferation genes.
prosthetic group Non-protein group, such as a metal atom or a haeme
group, associated with a protein and essential for its biological
function.
proteases Enzymes that catalyse the hydrolysis of peptide bonds, thus
digesting proteins. Also known as proteinases.
protein Heteropolymer (q.v.) of amino acids linked by peptide bonds.
May contain one or more polypeptide chains.
protofilament Element of microtubule structure made up of tubulin
dimer subunits.
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protoplast A plant cell with the cell wall removed, usually by treatment
with enzymes.
protoplast fusion The joining of two protoplasts to form a hybrid cell.
purine Double-ring base found in nucleic acids, the most common being
adenine and guanine.
pyrimidine Single-ring bases such as cytosine, thymine and uracil.
quaternary structure Level of protein structure in which two or more
subunits, each with its own secondary and tertiary structure, are held
together in a complex 3-D arrangement.
R-group Used to describe the variable functional group in amino acids.
Responsible for determining the properties of polypeptide chains.
receptor Protein to which a signalling molecule binds to elicit an
intracellular response.
recombinant DNA (rDNA) General term used to describe the
techniques of gene manipulation which generate DNA molecules with
sequences not found naturally.
residue Applies to the element of a monomer that is incorporated into a
polymer. Thus amino acids in proteins are called amino acid residues.
restriction endonuclease A nuclease that recognises a specific base
sequence in DNA (usually 4, 5 or 6 base pairs in length) and cuts
within or close to that sequence.
Rhizobium spp. Bacteria involved in nitrogen fixation, associated with
root nodules in certain plants.
ribosomal RNA (rRNA) RNA molecule that is a component of ribosome
structure. Occurs as different forms, characterised by their size. Cf.
messenger RNA, transfer RNA.
ribosome The ‘jig’ that brings together mRNA and charged tRNA
molecules during protein synthesis. Composed of a complex
arrangement of ribosomal RNA and ribosomal proteins
RNA Ribonucleic acid (infrequently the term ribosenucleic acid is used).
A polynucleotide, usually single-stranded, composed of
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GLOSSARY
ribonucleotides with the bases adenine, guanine, cytosine and uracil.
Occurs as different forms such as mRNA, rRNA and tRNA q.v. The
genetic material in some viruses.
sarcolemma The membranous sheath around a muscle fibre.
SCIDS Severe combined immunodeficiency syndrome; a disease caused
by a deficiency in the enzyme adenosine deaminase (ADA, q.v.). A
target for gene therapy.
second messenger A small molecule involved in signal transduction.
Generates a specific intracellular response to a signal received by a
transmembrane receptor. Examples of second messengers include
cyclic AMP (cAMP), inositol triphosphate (IP 3) and diacylglycerol
(DAG).
secondary structure In proteins, refers to the α-helix and β-sheet
arrangements of the polypeptide chain. May also be used for some
nucleic acid structures.
signal transduction The conversion of signals from one form into
another, as in the conversion of an extracellular signal to an
intracellular response, often via a receptor protein and a second
messenger q.v.
single-locus probe In DNA fingerprinting, refers to a probe that binds
to a single sequence in the genome. Diploid organisms will usually
show two bands in a profile, one of maternal and one of paternal
origin.
sodium-potassium pump Transmembrane protein complex that
‘pumps’ sodium out of the cell and potassium into the cell in a 3:2
ratio, using ATP hydrolysis to drive the process. Also known as the
Na +-K+ ATPase.
somatic cell Body cell, i.e. any cell in an organism apart from the
reproductive cells.
spindle fibres Microtubule-based fibres used to pull chromosomes or
chromatids apart during cell division.
spindle poles The ends of the spindle fibre structure not attached to
the centromere of the chromosome. Cf. centrosome, microtubule
organiser.
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GLOSSARY
starch Storage polysaccharide of plants, composed of glucose
monomers joined by α(1,4) bonds. May be unbranched (amylose) or
branched (amylopectin).
steroids Large group of polycyclic lipids based on a 4-ring structure.
Examples include cholesterol and testosterone.
subunit A component of a multi-subunit complex such as a large protein
or a ribosome.
synthase (synthetase) An enzyme that catalyses the joining of two
molecules.
tertiary structure Higher-order structure in proteins, where the
polypeptide is folded into a complex 3-D shape or conformation.
testosterone Steroid hormone that is the main male sex hormone in
mammals.
thymine Nitrogenous base found in DNA but not in RNA, where it is
replaced by uracil. Forms A:T base pairs with adenine.
thyroxine Iodine-containing hormone produced by the thyroid gland.
Derived from the amino acid tyrosine.
Ti plasmid Plasmid of Agrobacterium used for gene manipulation in
plants.
totipotent Having the potential to develop into all the types of cell that
are found in the adult organism.
transcription The synthesis of an RNA copy of DNA by the enzyme RNA
polymerase.
transfer RNA (tRNA) Clover-leaf shaped RNA which binds a specific
amino acid and has an anticodon which pairs with the codon in
mRNA. Cf. messenger RNA, ribosomal RNA.
transgenic An organism that has been genetically modified to carry a
‘foreign’ gene.
translation The synthesis of proteins, involving ribosomes, tRNAs with
their associated amino acids, and mRNA.
BIOLOGY
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GLOSSARY
transmembrane protein A protein that is embedded in a membrane so
that one portion is on the outside and one on the inside. Often
function as receptor proteins.
triacylglycerol A lipid composed of glycerol plus three fatty acids.
Often called triglycerides.
tricarboxylic acid (TCA) cycle Biochemical pathway for the complete
oxidation of pyruvate. Also known as the citric acid cycle or Krebs
cycle.
trypsin Protease, formed from its zymogen (q.v.) trypsinogen, involved
in digestion.
tubulin Protein component of microtubules q.v. Occurs as two forms, α
and β.
tumour-suppressor genes Also known as antiproliferation genes, these
are involved in controlling (restricting) cell division activity. When
defective, tumours may form.
uracil Nitrogenous base found in RNA only, where it replaces thymine.
Forms A:U base pairs with adenine.
vacuole Fluid-filled membrane-bound organelle in plant cells which
shows little structural detail. Often occupies a large proportion of the
cell volume.
X-ray crystallography Method of determining the structure of complex
molecules by analysing the diffraction patterns of X-rays by crystals of
the molecule.
zymogen Inactive precursor of some enzymes. Converted to the active
enzyme by cleavage of the polypeptide.
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